共表达FMDV P1-2A和3C重组鸡痘病毒疫苗与核酸疫苗的实验免疫研究

将口蹄疫病毒(FMDV)衣壳蛋白前体P1-2A基因和蛋白酶3C基因分别插入到鸡痘病毒表达载体质粒pUTA-16Lac Z的复合启动子和单一启动子的下游,构建共表达重组鸡痘病毒转移载体质粒pUTAL3CP1,与282E4株FPV共转染鸡胚成纤维细胞,经RT-PCR、IFA及Western blotting检测,成功获得能正确表达目的蛋白的重组鸡痘病毒株vU-TAL3CP1。并与FMDV重组核酸疫苗免疫小鼠实验表明,各实验免疫组均能诱导小鼠产生特异性的细胞免疫和体液免疫反应,其中以重组鸡痘病毒与核酸疫苗联合免疫效果好于其它实验各组。     

口蹄疫三价重组鸡痘病毒疫苗分子设计及其构建

针对我国口蹄疫(FMD)流行情况,利用计算机软件模拟分析,构建了口蹄疫病毒(FMDV)复合多表位基因工程疫苗表达盒OAAT.该表达盒以O型、A型FMDV结构蛋白VP1全基因和AsiaI型FMDV 2个基因拓扑型的结构蛋白VP1基因上的5个抗原表位基因(其中2个抗原表位来源于FMDVIND63/7株,该毒株与我国新疆分离株属于同一基因拓扑型,其余3个抗原表位来源于FMDVYNBS/58株)作为主要免疫原基因,以非结构蛋白上的2个Th2细胞表位基因及结构蛋白上的1个Th2细胞表位基因作为辅助基因.将构建好的表达盒OAAT和猪IL-18基因分别插入到鸡痘病毒(FPV)表达载体pUTA-16-LacZ复合启动子(ATI-P7.5×20)和单一启动子(P7.5)下游,构建了携带OAAT表达金和猪IL-18基因的重组鸡痘病毒转移载体质粒pUTAL-OAAT-IL18.应用脂质体转染法,将重组鸡痘病毒转移载体质粒与282E4株FPV共转染鸡胚成纤维细胞(CEF),经BrdU进行3次加压蚀斑筛选后,以不同代次的细胞mRNA为模板,利用FMDV VP1基因和猪IL-18基因特异引物进行RT-PCR和IFA检测,筛选出OAAT和猪IL-18基因共表达的重组鸡痘病毒rF-PV-OAAT-IL18,该重组鸡痘病毒的构建为新型FMDV活载体疫苗的研究奠定了基础.     

HIV-1中国流行株gp120基因在痘苗病毒中的表达及其与核酸疫苗的实验免疫研究

将HIV－1中国流行株gp120基因在痘苗病毒中进行表达,以期获得重组痘苗病毒,与核酸疫苗混合免疫,评价免疫效果,为艾滋病疫苗开发研制打下基础。将HIV－1中国流行株gp120基因片段插入到pJ38载体启动子下游,经同源重组和血凝素阴性空斑筛选重组痘苗病毒,SDS－PAGE、Western blot检测目的蛋白。以重组病毒和核酸疫苗免疫BALB／c小鼠,进行淋巴细胞转化实验、CTL、CD4＋CD8＋T细胞数目以及血清抗体等细胞免疫和体液免疫指标检测。结果获得的重组痘苗病毒vJ38pg120能够表达Gp120蛋白并诱导机体产生细胞免疫和体液免疫,具有良好的免疫原性,其免疫效果以2rVV-DNA混合方式为最好。重组痘苗病毒vJ38gp120。     

结核分枝杆菌DNA疫苗的免疫原性和保护效率的比较研究

对结核分枝杆菌两种DNA单价疫苗的免疫应答和保护效果进行了评价和比较。将结核分枝杆菌抗原蛋白。MPT70(22kDa)和PstS-3(40kDa)克隆到原核表达载体pET22b中,酶切和测序鉴定正确后转化入E．coli BL21(DE3)PLysS宿主菌株内中,通过诱导表达纯化获得目的蛋白用于DNA疫苗效价鉴定。用真核表达载体pJW4303构建了MPT70,PstS-3(包括信号肽的全基因)两种重组真核质粒(DNA疫苗),进行了酶切鉴定和序列分析,确定含有正确的读码框。用上述DNA疫苗分别肌注免疫小鼠,三免小鼠后21天MPT70和PstS-3抗体均达到为1：12800,三免后21天小鼠的脾脏细胞诱导产生较高水平的MPT70和PstS-3特异性的细胞因子γ-干扰素,浓度分别为16872．82pg／ml和11460．98pg／ml.三次免疫后经静脉强毒攻击,两组DNA疫苗免疫鼠的肺脏和脾脏的载菌数均比阴性对照组少,表明这两种疫苗都能诱导机体产生细胞免疫和体液免疫应答,而且以前者为主。综合各项指标发现,DNA疫苗MPT70要优于PstS-3。     

以融合蛋白形式在E·Coli中表达人神经生长因子

将人神经生长因子基因（hNGF）克隆到pMalC2质粒中，构建了表达产物为麦芽糖结合蛋白（MBP）与hNGF的融合蛋白重组质粒。经由内切酶再水解及DNA序列测定等分析表明，重组质粒含hNGF基因（360bP），该质粒在E·Coli中的表达产物为融合蛋白MBP－hNGF（55kd），光密度扫描表明，其表达量约为30％。     

人免疫缺陷病毒I型p24gag基因的重组与表达

将编码人Ｉ型免疫缺陷病毒（ＨＩＶ－１）衣壳蛋白的ｐ２４ｇａｇ基因片段，克隆到原核表达载体ｐＥＴ－１７ｂ的Ｔ７噬菌体启动子下游，构建成了重组表达质粒ｐＥＴ２４，并使ｐ２４ｇａｇ基因片段在大肠杆菌ＢＬ２１（ＤＥ３）中高效表达，产物为３０ｋＤａ的ｓ１９－ｐ２４融合蛋白，表达量占菌体总蛋白的３８．４％。重组ｐ２４蛋白均与抗ｐ２４单克隆抗体及ＨＩＶ－１阳性血清发生特异性反应，具有较好的抗原性。     

大肠杆菌热敏肠毒素和耐热肠毒素基因融合及其免疫原性研究

采用基因工程技术将编码大肠杆菌热敏肠毒素亚单位（ＬＴＢ）基因和耐热肠毒素（ＳＴ）基因进行体外重组,得到的融合基因能在大肠杆菌中表达。重组菌株免疫动物后,均能诱发产生抗ＬＴ和ＳＴ抗体。实验结果表明,ＬＴ／ＳＴ融合蛋白不仅保持了ＬＴＢ的免疫原性和与神经节甙酯ＧＭ１的结合能力,而且也赋予本来没有免疫原性的ＳＴ免疫原性,并极大地降低了ＳＴ的生物毒性,为构建理想的致腹泻大肠菌苗奠定了基础。     

高纯度白喉毒素突变体CRM197的制备及鉴定

为了制备高纯度的白喉毒素无毒突变体CRM197,可作为载体蛋白用于细菌性结合疫苗开发,对CRM197基因进行大肠杆菌密码子优化,并克隆至表达载体pET28a(+）中,将鉴定正确的重组质粒pET28a-CRM197转化到大肠杆菌BL21(DE3),经IPTG诱导表达,并分析其表达形式及表达条件的优化。再对表达的重组CRM197蛋白进行纯化,最后对纯化的CRM197进行WB、纯度、分子量和圆二色谱等检测项目的初步鉴定分析。PCR酶切和测序鉴定结果表明重组质粒pET28a-CRM197构建成功,将其转化到大肠杆菌BL21(DE3)中,获得重组工程菌(E.coli(DE3/p28a/197)。该重组工程菌发酵的最佳接种量为5%～10%(v/v),且主要以包涵体形式表达,收获的菌体在高压均质机破碎压力为1 000 bar的条件下进行破碎,然后利用6 mol/L盐酸胍进行溶解变性,复性及经30 kD超滤膜包超滤后上纯化系统AKTA pure150 M,经一步阴离子交换层析进行纯化,其纯度可达98%以上,WB、分子量及圆二色谱等鉴定结果均与标准品一致。综上所述,成功建立大肠菌表达系统CRM197制备...     

东亚钳蝎昆虫毒素BmKIT的cDNA序列分析

利用ＲＴ－ＰＣＲ方法从东亚钳蝎的尾腺总ＲＮＡ中扩增出挛缩型昆虫毒素ＢｍＫＩＴ的ＣＤＮＡ〈并对其核苷酸序列进行了分析。将含该基因的重组分泌型表达质料ＰＥｘＳｅｃＩ－ＢｍＫＩＴ转化大肠杆菌ＢＬ２１（ＤＥ３）,表达出分泌型的融合蛋白     

密码子优化的牛精蛋白基因在大肠杆菌中的表达

以PCR的方法得到牛精蛋白基因的基因，去其内含子，得到牛精蛋白cDNA，克隆入原核表达载体pGEX-2T中，组装成表达载体pGEX-2T-PE，利用大肠杆菌偏好的编码精氨酸的密码子CGT将野生型牛精蛋白基因中编码精氨酸的稀有密码子（AGA或AGG）替换掉，通过基因合成得到密码子优化的牛精蛋白的基因，将其克隆入原核表达载体pGEX-2T中，组装成表达载体pGEX-2T-PS。将这两个表达载体分别转化入大肠杆菌表达菌株BL21中，经IPTG诱导，同样条件下，野生型牛精蛋白基因无法得到表达，密码子优化的牛精蛋白基因能够得到良好的表达，表达产物约占细菌总蛋白的18％，将表达蛋白纯化，进行DNA－蛋白结合实验，发现其能与DNA发生非特异性的结合。     

Assembly of Immunogenic Protein Particles toward Advanced Synthetic Vaccines

Immunogenic carrier proteins such as the non-toxic diphtheria toxin variant, cross-reacting material 197 (CRM197), are widely used in subunit vaccine formulations to boost immunogenicity of chemically conjugated antigens. Conjugate vaccines are inherently expensive due to laborious manufacturing steps. Here, this work develops a particulate vaccine platform based on using engineered Escherichia coli to assemble CRM197-antigen fusion proteins into discrete submicron-sized particles. This approach enables precise loading of diverse antigens and epitopes enhancing their immunogenicity. A cost-effective, high-yield, and scalable biomanufacturing process is developed. Purified particulate CRM197-antigen vaccines are ambient-temperature stable. CRM197 particles incorporating pathogen-specific antigens or epitopes from SARS-CoV-2, Streptococcus pyogenes (group A), and Mycobacterium tuberculosis induced cell-mediated and humoral immune responses mediating protective immunity in respective animal models of infection. The CRM197 particle vaccine platform is versatile, enabling co-delivery of selected antigens/epitopes together with immunogenic CRM197 as discrete stable particles avoiding laborious manufacture of soluble CRM197 and antigen followed by chemical conjugation.     

鳗弧菌油乳化二价口服疫苗免疫养殖大菱鲆的免疫应答及免疫效果的研究

以鳗弧菌M3和SMP1为细菌抗原制备了油乳化二价疫苗,用饵料包埋后连续7天口服免疫大菱鲆鱼,评价鱼的免疫应答和疫苗的保护效果.结果显示,乳化疫苗在鱼后肠刺激产生的溶菌酶和抗蛋白酶活性以及抗体水平均高于未乳化疫苗(P＜0.05);并且在乳化疫苗免疫的鱼血清检测到明显升高的M3抗体效价(P＜0.05).原位杂交结果显示,乳化疫苗免疫鱼的后肠IgM的产生水平高于未乳化疫苗免疫鱼.攻毒实验显示,乳化疫苗免疫的鱼对M3和SMP1的感染分别获得100%和50%的免疫保护率,而未乳化疫苗获得的免疫保护率分别为57.9%和0%.结果表明,鳗弧菌油乳化二价口服疫苗能引起大菱鲆后肠的非特异性和特异性免疫应答并有效地抵抗鳗弧菌的感染,适合用作水产口服鱼用疫苗.     

重组质粒pcFlaA在青石斑鱼肌肉中的表达研究

在哈维氏弧菌TS-628菌株鞭毛丝蛋白FlaA基因末端加上一段编码Flag短肽的核苷酸序列作为检测标记后,将该基因克隆到真核表达载体pcDNA3.1( ),酶切、PCR扩增及重组质粒测序证实基因片段插入正确,将该重组质粒命名为pcFlaA.将pcFlaA以肌肉注射方式免疫青石斑鱼.免疫后第7天开始检测鞭毛丝蛋白在石斑鱼肌肉中的表达状况,之后每隔1周检测1次,共检测4次.首先采用PCR技术在DNA水平检测重组质粒转染石斑鱼肌肉细胞的情况,再以RT-PCR法在mRNA水平上检测转染质粒在鱼肌肉中的转录,最后以免疫组化染色技术在蛋白质水平上检测目的蛋白的表达.结果在DNA及mRNA水平上均可检测到目的条带,在蛋白质水平上可检测到明显阳性位点,由此证实pcFlaA可以转染石斑鱼肌肉细胞并可在其中进行表达,而且质粒在鱼体内持续表达的时间至少1个月.     

水产养殖动物核酸疫苗的研究与应用现状

介绍了鱼用核酸疫苗载体的构建、诱导的免疫反应机制、接种途径、佐剂的免疫促进作用及安全性等研究现状。     

激光光散射法表征人用皮卡狂犬病疫苗分子量与人体副反应关系的观察

为了解人用皮卡狂犬病疫苗分子量与人体副反应的关系，用激光光散射法进行了实验。结果表明：当疫苗中皮卡浓度为１１．９５ｍ ｇ／ｍ ｌ时，其重均分子量Ｍ ｗ ＝ ２９．６×１０４，２０名志愿者中７人出现局部和全身过敏反应；当疫苗中皮卡浓度为１ｍ ｇ／ｍ ｌ时，其重均分子量Ｍｗ ＝ ５．６×１０４，２０名志愿者中无一人出现接种后副反应。这说明，用激光光散射法检测人用皮卡狂犬病疫苗分子量的大小与人体副反应的关系密切。     

Distribution of vaccine/antivirals and the ‘least spread line’ in a stratified population

We describe a prioritization scheme for an allocation of a sizeable quantity of vaccine or antivirals in a stratified population. The scheme builds on an optimal strategy for reducing the epidemic''s initial growth rate in a stratified mass-action model. The strategy is tested on the EpiSims network describing interactions and influenza dynamics in the population of Utah, where the stratification we have chosen is by age (0–6, 7–13, 14–18, adults). No prior immunity information is available, thus everyone is assumed to be susceptible—this may be relevant, possibly with the exception of persons over 50, to the 2009 H1N1 influenza outbreak. We have found that the top priority in an allocation of a sizeable quantity of seasonal influenza vaccinations goes to young children (0–6), followed by teens (14–18), then children (7–13), with the adult share being quite low. These results, which rely on the structure of the EpiSims network, are compared with the current influenza vaccination coverage levels in the US population.     

脂质体对牙鲆淋巴囊肿病毒核酸疫苗免疫活性的影响

将淋巴囊肿病毒核酸疫苗pEGFP-N2-LCDV0.6kb用脂质体梭华-SoftastTM包裹后肌肉注射入牙鲆体内,通过测定牙鲆外周血、肠、脾脏和前肾的淋巴细胞增殖反应、呼吸爆发活性以及抗体产生水平,评价脂质体对pEGFP-N2-LCDV0.6kb免疫活性的影响.结果表明,脂质体可显著增强该核酸疫苗的免疫活性和提高保护率,可作为一种有应用前景的核酸疫苗佐剂.     

Applications of polymeric adjuvants in studying autoimmune responses and vaccination against infectious diseases

Polymers as an adjuvant are capable of enhancing the vaccine potential against various infectious diseases and also are being used to study the actual autoimmune responses using self-antigen(s) without involving any major immune deviation. Several natural polysaccharides and their derivatives originating from microbes and plants have been tested for their adjuvant potential. Similarly, numerous synthetic polymers including polyelectrolytes, polyesters, polyanhydrides, non-ionic block copolymers and external stimuli responsive polymers have demonstrated adjuvant capacity using different antigens. Adjuvant potential of these polymers mainly depends on their solubility, molecular weight, degree of branching and the conformation of polymeric backbone. These polymers have the ability not only to activate humoral but also cellular immune responses in the host. The depot effect, which involves slow release of antigen over a long duration of time, using different forms (particulate, solution and gel) of polymers, and enhances the co-stimulatory signals for optimal immune activation, is the underlying principle of their adjuvant properties. Possibly, polymers may also interact and activate various toll-like receptors and inflammasomes, thus involving several innate immune system players in the ensuing immune response. Biocompatibility, biodegradability, easy production and purification, and non-toxic properties of most of the polymers make them attractive candidates for substituting conventional adjuvants that have undesirable effects in the host.     

Vaccine delivery: Nanopatch‐Targeted Skin Vaccination against West Nile Virus and Chikungunya Virus in Mice (Small 16/2010)

The ‘Nanopatch’ (NP) comprises arrays of densely packed projections with a defined geometry and distribution designed to physically target vaccines directly to thousands of epidermal and dermal antigen presenting cells (APCs). These miniaturized arrays are two orders of magnitude smaller than standard needles—which deliver most vaccines—and are also much smaller than current microneedle arrays. The NP is dry‐coated with antigen, adjuvant, and/or DNA payloads. After the NP was pressed onto mouse skin, a protein payload co‐localized with 91.4 ± 4.1 APC mm⁻² (or 2925 in total) representing 52% of the delivery sites within the NP contact area, agreeing well with a probability‐based model used to guide the device design; it then substantially increases as the antigen diffuses in the skin to many more cells. APC co‐localizing with protein payloads rapidly disappears from the application area, suggesting APC migration. The NP also delivers DNA payloads leading to cutaneous expression of encoded proteins within 24 h. The efficiency of NP immunization is demonstrated using an inactivated whole chikungunya virus vaccine and a DNA‐delivered attenuated West Nile virus vaccine. The NP thus offers a needle‐free, versatile, highly effective vaccine delivery system that is potentially inexpensive and simple to use.     

DDA提高结核杆菌组合DNA疫苗免疫效果的研究

将编码结核杆菌的三种抗原Ag85B,MPT64,MPT83的基因片段分别插入到真核表达载体中,混合后作为组合疫苗免疫小鼠,DDA作为佐剂提高了三价疫苗的免疫原性和免疫保护效果.添加DDA后Ag85B,MPT64,MPT83抗原特异的IFN-γ的含量分别为(265.37±79.2)U/ml,(185.31±58.3)U/ml,(108.13±54.4)U/ml,分别比非佐剂组高16U/ml,45U/ml,2 U/ml.IL-4的含量在各组中相差不多,仅为纳克级.攻毒后细菌计数结果显示,肺脏和脾脏的载菌量在添加佐剂的三价组中降低了三个数量级,都达到了未加佐剂组相应脏器的一半菌量.病理切片显示结果与载菌量一致,添加佐剂组肺部淋巴细胞相对较少,巨噬细胞增多.因此,DDA作为佐剂提高了组合疫苗的免疫效果.