Sensitization to Sitophilus granarius in selected suburban population of South Poland

Storage insects may cause occupational allergies in humans. The identification of the prevalence of IgE reactions were measured from protein fractions prepared from multiple life stage of granary weevil Sitophilus granarius [SG] is reported. Sera of 30 patients from a suburban population of Upper Silesia (South Poland) were tested for the presence of IgE antibodies to antigens from larvae, pupae and adults of both sexes of the beetle. To identify protein fractions containing potential allergens, proteins collected from four life stages of granary weevil were fractionated by SDS-PAGE and probed with anti-human, anti-IgE monoclonal antibodies. The proteins were fractionated by SDS PAGE and identified by Western blot. The patients’ antibodies against particular antigens were identified using anti-human anti-IgE monoclonal antibody. The conducted immunological analysis showed the existence of many protein fractions for each life stage of SG which give positive reactions with IgE antibodies. The largest number of allergenic potential fractions was shown in pupae (60 protein fractions) while the smallest amount was shown in larvae (44 protein fractions). Summarizing, the obtained results suggest the existence of many protein fractions with an allergenic potential multiple life stages of SG. This indicates that all developmental stages of SG may be a serious source of antigens and potential risk factors for the exposed persons.     

Why are some proteins allergenic? Implications for biotechnology

In recent years, a number of agricultural crops have been developed with recombinant DNA technology. Because the transferred genes code for proteins that are ordinarily not present in these particular foods, there is concern about the potential allergenicity of these new crop varieties. Foods contain many proteins; however, only a small fraction are allergens. Although the structural properties of proteins that cause allergic reactions have not been characterized completely, known food allergens in general have molecular weights between 10 and 70 kDa, stimulate the immune response (induce the production of allergen‐specific IgE), and are stable molecules that are resistant to processing, cooking, and digestion. Although any type of food is potentially allergenic, the majority of food allergies are caused by a small group of foods (cows’ milk, nuts, legumes, eggs, seafood). Cross‐reactivities occur within a given food group and between foods and seemingly unrelated proteins.

Even though most transgenic foods are considered safe, biotechnological manipulation can affect crop allergenicity. The safety evaluation of transgenic foods is relatively easy when the allergenicity of the gene sources are known. The recombinant food can be assayed using traditional in vitro inhibition assays. Recently, reduced allergen content of biotechnologically altered rice was shown. In contrast, increased allergenicity was demonstrated in transgenic soybeans after a methionine‐ and cystine‐rich protein from Brazil nuts, identified as a major Brazil nut allergen, was expressed in soybean to increase its content of sulfur‐rich amino acids. The most difficult issue regarding transgenic food allergenicity is the effect of transfer of proteins of unknown allergenicity. The challenge is to determine whether these proteins are allergenic as there is no generally accepted, established, definitive procedure to define or predict a protein's allergenicity. Comparing the structures of the transferred protein with known allergens and allergen epitopes could be one approach. Additionally, Th‐2 cell stimulation, the production of IL‐4, and IgE antibody induction in animal models may help to evaluate the potential allergenicity of a protein.

In conclusion, there is no evidence that recombinant proteins in newly developed foods are more allergenic than traditional proteins. The evidence suggests that the vast majority of these proteins will be completely safe for the consumer. The concern is that if a few transgenic foods cause serious allergic reactions, this could undermine the public's confidence in such products. It is essential that proper guidelines are established and tests are developed to assure that this will not occur.     

Prevalence of sensitization to extracts from particular life stages of the saw-toothed grain beetle (Oryzaephilus surinamensis) in citizens of selected suburban areas of Southern Poland

Many species of insects are agricultural pests which cause not only economic losses but also allergies in humans. The subject of this study was to identify important antigens from the saw-toothed grain beetle – Oryzaephilus surinamensis [OS]. Sera of 30 patients from a suburban population of Upper Silesia (Southern Poland) were tested for the presence of IgE antibodies to antigens from particular active life stages of OS (larvae, pupae and adults of both sexes). The collected proteins were fractionated by SDS PAGE and identified by Western blot. The patient's antibodies against particular antigens were identified using anti-human anti-IgE monoclonal antibody. The conducted studies showed the existence of many protein fractions for each life stage of OS which give positive reactions with IgE antibodies. The largest number of allergenic potential fractions was shown in females (23 protein fractions) and pupae (22 protein fractions) while smaller amount was shown in larvae (18 protein fractions) and males (14 protein fractions). Majority of the sera (25/30) showed positive reactions to protein fractions 25–29 kDa and 30–34 kDa from pupae of OS. The obtained results may indicate the existence of many protein fractions with an allergenic properties in OS. It also should be stressed that all life stages of this storage insect may provoke allergic reactions in exposed subjects.     

Allergenic responses of red kidney bean (Phaseolus vulgaris cv chitra) polypeptides in BALB/c mice recognized by bronchial asthma and allergic rhinitis patients

Allergenicity potential of red kidney beans (Phaseolus vulgaris cv chitra) was assessed and attempts were made to identify the responsible proteins by pepsin digestibility assay and IgE immunoblotting. To evaluate allergenic potential, BALB/c mice were sensitized with red kidney bean proteins and levels of specific immunoglobulin, histamine, mast cell protease-1, cytokines and CCL-2 were measured. To confirm our findings in BALB/c, the studies were also extended to human subjects. Human sera collected from control subjects and allergic patients after skin prick test were used for IgE immunoblotting, measuring the levels of total and specific IgE and determining cross reactivity of red kidney bean with other legumes. Red kidney bean allergenic potential was evident by significant increase in specific IgE, IgG1, histamine, mast cell protease-1 and Th2 cytokine levels in comparison to control. Enhanced level of eosinophils in jejunum, prominent anaphylactic symptoms, and eruptive histopathological changes give indication towards red kidney beans allergenicity. IgE immunoblotting detected five protein components with molecular weights of approximately 170, 100, 43, 34 and 20 kDa. Red kidney bean proteins showed cross reactivity with peanut, soybean, chickpea and black gram. Finally, this work demonstrated that red kidney beans may induce allergic response in mice similar to human subjects, with identification of five clinically relevant allergenic protein components.     

Allergy assessment of foods or ingredients derived from biotechnology, gene-modified organisms, or novel foods

The introduction of novel proteins into foods carries a risk of eliciting allergic reactions in individuals sensitive to the introduced protein and a risk of sensitizing susceptible individuals. No single predictive test exists to perform a hazard assessment in relation to allergenic properties of newly expressed proteins in gene-modified organisms (GMOs). Instead, performance of a weighted risk analysis based on the decision tree approach has been suggested. The individual steps of this analysis comprise sequence homology to known allergens, specific or targeted serum screens for immunoglobulin E (IgE) cross-reactions to known allergens, digestability studies of the proteins in simulated gastric and/or intestinal fluids, and animal studies. These steps are discussed and five examples of risk evaluation of GMOs or novel foods are presented. These include ice-structuring protein derived from fish, microbial transglutaminase, GMO-soybeans, amylase and the Nangai nut.     

Assessment of allergic potential of (novel) foods

In safety assessment of Novel Foods such as functional foods, allergy is a special issue on which particular emphasis has been placed. The reason for such concern is that incidence of food allergies is constantly and rapidly increasing. The severity of the reported incidents and the number of foods incriminated are also on the rise. The outstanding challenge is to understand what makes a common innocuous protein or peptide behave as an allergen for some groups of people, or why it may suddenly or progressively become a much more potent allergen than usual. It is therefore necessary to consider the risks of creating or unmasking new immunoreactive structures, or of overexposure to already reactive substances, as a result of new food-production and processing technologies. No test such as the use of animal models, the analysis of structure, function and physico-chemical properties is as yet available to evaluate or predict the allergenicity of a “novel” protein in a wholly reliable and objective manner. No indication has yet suggested that novel foods, and particularly recombinant proteins or genetically modified foods, would be more (or less) allergenic than the corresponding conventional foods. No particular structure can be described as being solely and intrinsically allergenic. The predictive approaches to determining the allergenic potential of NFs should therefore be subject to case-by-case critical appraisal allied to mandatory implementation of monitoring of the potential postmarketing impact of these new foodstuffs on public health.     

Development of a Biological Assay to Determine the Allergenic Potential of Foods

Food allergies represent a risk for many people in industrialized countries. Unrecognizable allergenic proteins of foodstuffs may be present as ingredients that are not labeled or as unknown cross-contamination. Such hidden allergens can cause severe reactions in allergics, even at minute quantities, sometimes with fatal outcome. For the verification of the presence of allergenic food constituents, analytical methods such as ELISA and PCR have been developed. However, these tests cannot measure allergenic potential. For this reason, a test system that measures the biological activity of allergens has been developed. It is based on the cellular mechanisms of the type I allergy. Rat basophilic leukemia cells (RBL-2H3) were transfected with the genes of the human high affinity receptor for IgE. The resulting cell line expressed the human receptor α-chain and could bind allergenspecific IgE from allergic subjects, in contrast to the parent cell line. After cross-linking of receptor-bound, allergen-specific human IgE by allergens, the cells released measurable inflammatory mediators. These cells were used for the analysis of a variety of allergen extracts, including extracts prepared from foods containing allergenic hazelnut and peanut. The comparative validation with existing ELISA and PCR for hazelnut and peanut demonstrated similar sensitivity and specificity. The established cell line will be a novel tool in the detection of allergens in complex mixtures, especially to address the issue of their allergenic potential, which cannot be accomplished by classical analytical methods. This will add valuable information about the allergenic potential of food constituents to the risk assessment of foods.     

Peanut polyamines may be non‐allergenic

Polyamines such as putrescine, spermidine and spermine have been implicated in preventing food allergies in early life, but they have also been reported to be able to bind to immunoglobulin E (IgE) antibodies in vitro (ie they are possibly allergenic). The objective of this study was to determine if polyamines bind in vitro to IgE antibodies from a pooled serum of five peanut‐allergic individuals. Levels of polyamines were also determined by ion‐exchange chromatography. Indirect and inhibition enzyme immunosorbent assays (ELISAs) were used to determine the IgE binding or allergenic properties of polyamines. Results showed that, of the three polyamines, spermidine was predominant in peanuts. In both indirect and inhibition ELISAs, IgE antibodies did not bind to the polyamines. It was concluded that polyamines from peanuts, unlike peanut proteins, are not allergenic or an additional threat to patients who are allergic to peanuts. Published in 2005 for SCI by John Wiley & Sons, Ltd.     

苦荞过敏原TBa 的表位突变及免疫活性鉴定

前期研究曾分离获得一种纯度均一的天然苦荞过敏蛋白(tartary buckwheat allergen a,TBa ),免疫检测发现其为苦荞中的主要过敏原。本实验采用定点突变的方法对得分较高的表位E1 的基因进行分子改造,构建5 个突变体L39R、L42R、L47R、V52R、L54R。在大肠杆菌BL21 中分别进行表达,经Ni2+-NTA 亲和纯化后得到纯度较高的重组突变体蛋白。ELISA 和点杂交检测发现,与突变前的E1 相比,突变体L42R、L47R、L54R 的免疫活性明显降低,这表明Leu42、Leu47、Leu54 在TBa 的过敏性中起着至关重要的作用。     

Requirements for effective IgE cross-linking on mast cells and basophils

This article reviews the characteristics of high affinity IgE receptors (FcepsilonRI) and their role in the response to allergenic proteins. The requirements for successful cross-linking of FcepsilonRI on basophils and mast cells and subsequent degranulation by allergenic proteins will be explained in detail. Methods for in vitro analysis of allergen-induced mast cell and basophil degranulation will be described and issues/problems in applying these methods will be discussed. Finally, implications for manipulation of protein allergens will be discussed.     

Germin-like protein Cit s 1 and profilin Cit s 2 are major allergens in orange (Citrus sinensis) fruits

Oranges are clinically relevant allergenic foods. To date, orange allergens have not been characterized in detail. The study is aimed at analyzing the sensitization profile in orange-sensitized subjects with and without clinical allergy, and to identify orange allergens. Fifty-six sensitized subjects with self-reported reactions to orange were grouped into reactors (anaphylaxis or multiple episodes of immediate reactions and/or positive challenge tests) and non-reactors (negative open food challenge tests). Allergens were characterized by IgE immunoblotting, N-terminal sequencing, IgE-inhibition assays, and mediator release assays were performed to determine the allergenic potency of orange profilin. Of 56 subjects, 23 were classified as orange allergic showing mainly an oral allergy syndrome. Of 23 subjects classified as orange allergic, 22 were sensitized to profilin, Cit s 2. In patients with mono-sensitization to profilin in vitro histamine releases up to 75% from basophils were induced using orange extract and purified plant profilins. Of the allergic patients 78% were sensitized to germin-like protein, Cit s 1. Both allergens showed retained IgE reactivity in heat-processed orange juice. Interestingly, subjects with and without clinical allergy showed a comparable sensitization profile. Profilin and germin-like proteins are major orange allergens. The potential clinical relevance of orange profilin was indicated by its strong capacity to release histamine from basophils. However, a predominant sensitization to both allergens in subjects without symptoms also indicates a high frequency of clinically insignificant sensitization.     

In silico predictability of allergenicity: from amino acid sequence via 3-D structure to allergenicity

In relation to the prediction of allergenicity three aspects have to be discussed: IgE immunogenicity, IgE cross-reactivity, and T-cell cross-reactivity. IgE immunogenicity depends largely on factors other than the protein itself: the context and dose and "history" of the protein by the time it reaches the immune system. It is, therefore, not fully predictable from structural information. In contrast, IgE cross-reactivity can be much more reliably assessed by in-silico homology searches in combination with in vitro IgE antibody assays. The in-silico homology search is unlikely to miss potential cross-reactivity with sequenced allergens. So far, no biologically relevant cross-reactivity at the antibody level has been demonstrated between proteins without easily demonstrable homology. T-cell cross-reactivity is much more difficult to predict than B-cell cross-reactivity. Moreover, its effects are more diverse. Yet, pre-existing cross-reactive T-cell activity is likely to influence the outcome not only of the immune response, but also of the effect phase of the allergic reaction. The question of whether any antigen can be allergenic is still a matter of debate.     

Digestibility and Immunoreactivity of Shrimp Extracts Using an In Vitro Digestibility Model with Pepsin and Pancreatin

Shellfish allergy affects 2% of the adult population in the United States. Identification of allergenic shrimp proteins is needed for improved management and assessment of shrimp allergy. We determined the temporal pepsin and pancreatin stability of total shrimp proteins using simulated physiological digestive conditions in vitro. Gel electrophoresis was used to determine protein stability, whereas immunoreactivity of protease stable proteins was determined using rabbit antigen‐specific antibodies. Potential allergenicity of protease stable proteins was determined utilizing human sera from shrimp allergic patients. Total shrimp myofibrillar proteins were pepsin‐ and pancreatin‐stable for up to 1 h after initiating digestion, whereas only pancreatin‐stable total shrimp proteins were Immunoglobulin G (IgG) immunoreactive. However, shrimp proteins of 32 and 25 kDa were pepsin and/or pancreatin stable and Immunoglobulin E (IgE) reactive, denoting the stability and potential allergenicity. These findings suggest that this in vitro digestibility model may be useful for the identification of shrimp allergenic proteins that are more resistant to physiologic digestive conditions and may elicit an immunologic response in vivo.     

苦荞过敏蛋白全长基因的克隆、表达及免疫学活性研究

荞麦属于一种常见的植物性过敏原。本实验在以往研究的基础上,以苦荞种子总RNA 为模板,通过RTPCR和5'-RACE 等方法,扩增、克隆获得苦荞过敏蛋白全长cDNA 序列。分析表明,该序列编码一个由515 个氨基酸残基组成的功能蛋白,并与甜荞过敏性贮藏蛋白的氨基酸序列同源性达到90% 以上。经原核表达及一步亲和纯化后得到纯度较高的重组苦荞过敏蛋白(rTBt)。采用竞争性ELISA 对其免疫活性分析显示,目的蛋白与荞麦过敏病人血清中的IgE 抗体可以特异结合。     

Production of pure protein and antibodies and development of immunoassays to detect Ara h 3 levels in peanut varieties

Peanuts are one of the most allergenic foods and are widespread in western food products. Therefore, there has been intense research into the allergic nature of the proteins involved. Ara h 3 is one of three immunodominant allergenic proteins. It is a 60‐kDa protein, which forms following cleavage of the preprotein and association of the resultant 40‐ and 20‐kDa subunits. The large subunit has been shown to harbour most of the reactive epitopes and has the protein fold likely responsible for its trypsin inhibitor activity. In this work, we have developed a method for the high‐level expression and purification of recombinant Ara h 3 40‐kDa subunit. Specific antibodies have been produced and applied to the secondary and tertiary screens of hundreds of peanut cultivars. Several of these cultivars were identified that have significantly reduced accumulation of Ara h 3.     

Structural conformers produced during malaria vaccine production in yeast

A recombinant protein expression system based on Saccharomyces cerevisiae has been used to express malarial vaccine candidate antigens. The antigens so produced have been used in three Phase 1 clinical trials and numerous preclinical non-human primate trials. Further Phase I trials are planned using these candidate vaccine antigens. These molecules were identified as attractive candidates for antimalarial vaccines, as they are all surface-exposed at some stage in the parasite's life cycle. They all share an unusual structural feature: epidermal growth factor (EGF)-like motifs. When these proteins are expressed in our S. cerevisiae expression system, they are produced as a series of stable structural conformers, each with a different disulphide bonding pattern. This leads to both biochemical and, more importantly, antigenic differences between the conformers (e.g. presence or absence of an antibody B cell epitope). These findings have important ramifications for other EGF-domain-containing proteins expressed in S. cerevisiae, or for proteins which contain other cysteine-folding motifs not normally expressed by this organism, both for vaccine production or for research/reagent purposes.     

Evaluation of the potential allergenicity of the enzyme microbial transglutaminase using the 2001 FAO/WHO Decision Tree

All novel proteins must be assessed for their potential allergenicity before they are introduced into the food market. One method to achieve this is the 2001 FAO/WHO Decision Tree recommended for evaluation of proteins from genetically modified organisms (GMOs). It was the aim of this study to investigate the allergenicity of microbial transglutaminase (m-TG) from Streptoverticillium mobaraense. Amino acid sequence similarity to known allergens, pepsin resistance, and detection of protein binding to specific serum immunoglobulin E (IgE) (RAST) have been evaluated as recommended by the decision tree. Allergenicity in the source material was thought unlikely, since no IgE-mediated allergy to any bacteria has been reported. m-TG is fully degraded after 5 min of pepsin treatment. A database search showed that the enzyme has no homology with known allergens, down to a match of six contiguous amino acids, which meets the requirements of the decision tree. However, there is a match at the five contiguous amino acid level to the major codfish allergen Gad c1. The potential cross reactivity between m-TG and Gad c1 was investigated in RAST using sera from 25 documented cod-allergic patients and an extract of raw codfish. No binding between patient IgE and m-TG was observed. It can be concluded that no safety concerns with regard to the allergenic potential of m-TG were identified.     

Allergen analysis and authenticity control: Development of mass spectrometry-based methods for animal-derived food products

Food allergy and food fraud involving animal-derived products are two of the most significant issues in food markets. On one hand, immunoglobulin E (IgE)-mediated allergic reactions after ingestion of fish, crustaceans, eggs, or milk are among the most prevalent and can happen even after ingestion of trace amounts. On the other hand, new rules regarding product commercialization (e.g., novel food regulation) are more and more created while fraudulent species substitution in fishery products is very common. Sensitive and accurate analytical methods for allergen quantification and species identification in commercial food products are therefore urgently required whether to help food industries inform allergic consumers, to ensure the food compliance with new regulations or to combat food fraud. In the past few years, bottom-up proteomic techniques, which rely on the detection of peptide biomarkers resulting from a tryptic digest of food proteins using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), have been emerging in this field. The selection of reliable allergen-specific or species-specific peptide biomarkers is one of the most crucial steps when developing such methods whether for qualitative protein detection (i.e., screening analysis) or protein absolute quantification. The first part of this dissertation relates therefore to the selection of allergen peptide biomarkers for fish, invertebrates, eggs, and milk in an experimental way using a single chaotropic urea extraction buffer. The allergenic proteins responsible for those severe reactions are mainly parvalbumin, tropomyosin, ovalbumin, and caseins. The protein extraction was first assessed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the major allergens were well extracted. After that, data-dependent MS/MS spectra which were obtained using digest samples of authentic animals or pure allergenic ingredients were processed against a matching protein database, and identified peptides were filtered according to several criteria such as the sequence length, amino acid composition, specificity, reproducibility, and sensitivity. Myosin proteins were also a target of choice for fish detection due to their high prevalence and sequence homology even if they are non-allergenic. Contrary to fish and invertebrate databases which included entries for only one specific protein (i.e., parvalbumin, myosin, or tropomyosin), egg and milk databases contained all known allergenic proteins or even additional proteins for egg yolk. Two extra selection steps were achieved for egg and milk markers based on more stringent criteria regarding their sensitivity after targeting them in the corresponding allergenic ingredients. At this point, 17, 13, 10, 10 and 12 potential markers were respectively selected for fish, invertebrates, egg white, egg yolk and milk. An alignment algorithm was used for all those markers to get an idea about their biological specificity. The biological specificity was verified experimentally for fish and invertebrate potential markers by targeted analysis in digest samples of animal species that are relevant in the food industry (vertebrates and invertebrates). This verification was not done for egg and milk markers as it was not regarded as significant due to reported cross-reactivity among avian eggs and among mammalian milk. Two potential parvalbumin markers were detected in other nonfish vertebrates, while all potential tropomyosin markers except one were specific to at least an invertebrate class belonging to the same phylum. Parvalbumin and myosin markers as well as tropomyosin markers were exclusively found in vertebrates or invertebrates. Marker detectability was checked by analyzing processed fish products as well as cooked fish for potential parvalbumin and myosin markers, while commercial insect-based food products such as cereals bars or pasta were studied for potential tropomyosin markers. All expected fish and invertebrate markers were detectable in those complex food products. Detectability of egg and milk markers was assessed by analysis of bread, cookies, and chocolate samples contaminated at different stages of the sample preparation with trace amounts (100 pg/g) of eggs and milk (i.e, fortified, spiked, and incurred digest samples). Two egg white markers and seven milk markers were detected in all those samples. The most suitable markers in terms of sensitivity and specificity were finally chosen for each allergenic product. Thus, besides two myosin fish global markers, five parvalbumin markers were retained including at least one of the investigated fish species. In addition, five tropomyosin markers were chosen, their specificity allowing us to distinguish crustacean tropomyosin from that of insects/arachnids, or mollusks. At last, two ovalbumin markers and three casein markers were confirmed to be the most suitable allergen markers respectively for egg white and milk. All those retained markers could be compiled in a single multiplex method. The automation of the sample preparation could also be a promising improvement whether for qualitative or quantitative analysis.     

Mitigation of peanut allergenic reactivity by combined processing: Pressured heating and enzymatic hydrolysis

Among food allergens, peanut is one of the most critical. This study evaluates peanut allergenic features after the combination of heat, pressure, and enzymatic digestion under sonication, by immunodetection using serum IgE of sensitized patients and mass-spectroscopy. In the studied population, there was a predominance of patients with sensitization to Ara h 9 (nsLTP) followed by sensitization to seed storage proteins (Sprot, Ara h 1, 2, 3, and 6). The Sprot sensitized patients showed higher reactivity. The enzyme E5 was efficient for inducing protein fragmentation and allergenic reactivity reduction when it was used combined with pressured heating treatments such as autoclave and Controlled Instantaneous Depressurization (DIC). Only a few Ara h 1 and Ara h 3 peptides were identified after enzymatic digestion of DIC peanut samples. The combination of pressured heating treatments and enzymatic hydrolysis was the most efficient method to strongly mitigate or even eliminate the allergenic potential of peanut. Our findings set a possibility for a group of patients in which their allergy could be treated with a processed less-allergenic peanut and consequently less risky, more easy and quicker desensitization treatment.Industrial relevanceThe findings identify innovative thermal, pressure and enzymatic processing conditions highly effective to mitigate or even abolish the allergenic potency of peanut, which may be relevant for consumers, clinicians, regulatory agencies and the food industry. The applications of processed peanut with reduced IgE binding potency for tolerance induction might be a convenient strategy.     

重组乳酸乳球菌滴鼻免疫小鼠后粘膜免疫特性及耐

乳酸菌NICE(乳链菌肽控制表达nisin controlled expression,NICE)系统可将治疗性蛋白或保护性抗原蛋白表达于细胞外或锚定于菌体的肽聚糖上,递呈抗原蛋白并激活粘膜免疫系统,刺激特异性s-IgA的产生使其作为粘膜免疫原递呈的活载体成为可能。本试验将构建的重组乳酸乳球菌PNZ8149/NZ3900-M/PRRS鼻腔免疫BALB/c小鼠,检测呼吸道粘膜中抗体s-IgA和血清中特异性抗体IgG含量,评价其动态变化情况,同时检测脾脏细胞因子IL-4和IL-10的活性。结果重组乳酸乳球菌PNZ8149/NZ3900-M/PRRS免疫小鼠后,在测定时间内重组菌试验组小鼠呼吸道粘膜中特异性s-IgA水平高于对照组,差异极显著((P<0.01);血清中特异性抗体IgG水显著高于对照组((P<0.01);脾脏细胞悬液中的IL-10和IL-4含量与对照组无差异性(p>0.05),结果表明重组菌PNZ8149/NZ3900-M/PRRS经粘膜途径免疫小鼠后能能刺激小鼠粘膜特异性抗体s-IgA和血清中特异性抗体IgG的分泌,且能避免粘膜免疫耐受的产生,为该重组疫苗的进一步应用奠定了理论基础。