FLP介导的酿酒酵母rDNA多拷贝定点整合

构建了一个带有ＦＲＴ位点以及启动子缺陷的ＵＲＡ３ｄ选择标记的环状质粒,并通过位点特异性重组将其整合到酵母染色体ｒＤＮＡ中放置的一个ＦＲＩ位点上。Ｓｏｕｔｈｅｒｎ杂交分析表明若处于很强的选择压力下,该质粒的拷贝数会发生有效的扩增。为了证明该质粒能够作为表达载体,将乙肝融合表面抗原ＳＡ－２８基因的表达单元插入该质粒后整合到ｒＤＮＡ位点。与ｒＤＮＡ位点整合了单个表达单元的菌株相比,ＳＡ－２８蛋白的表达量     

人GM－CSF在链霉菌中的表达

报道了ＰＩＪ４５９／ＧＭ－ＣＳＦ基因表达质粒的构建，以及该重组质粒在变铅青链霉菌Ｓ．ｌｉｖｉｄａｎｓＴＫ５４中的表达，结果表明：ＰＩＪ４５９／ＧＭ－ＣＳＦ质粒具有表达ｈＧＭ－ＣＳＦ的功能。重组菌株细胞裂解液经ＴＦ－１细胞培养及集落形成实验均证实了其生物学活性。并分析了影响外源基因表达的某些因素，如ＳＤ序列与ＡＴＧ之间的距离，时间对表达的诱导等，探讨了外源基因高效表达的适宜条件。     

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.     

植物抗体基因工程：Ⅲ．马铃薯Y病毒小鼠中和抗体链基因的序列分析及其植物表达载体的构建

序列分析表明，马铃薯Ｙ病毒小鼠中和抗体轻链基因（Ｋ６）全长为９５６个核苷酸碱基（不包括ｐｏｌｙ（Ａ）尾巴），其中包括５＇端非编码区３１个核苷酸碱基，编码轻链信号肽的５７个核苷酸碱基，编码成熟蛋白的６５７个核苷酸基和３＇端非编码区的２１１个核苷酸碱基。与已知的其它Ｋ轻链基因（ＭＲＫ１６）相比，核苷酸的序列同源性为９８．７％，由核苷酸序列所推导出的氨基酸序列同源性为９７．０％，其变异主要发生在三个抗原识别位点内（ＣＯＲ）。将完整的抗体轻链基因正向插入植物表达载体ｐＢ１２１中的原ＧＵＳ基因位置上，置于花椰菜花叶病毒３５Ｇ启动子控制之下，获得了能够在植物细胞内表达马铃薯Ｙ病毒中和抗体轻链基因的植物表达载体ｐＹＬ。     

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。     

外源性野生型p53基因对人喉癌等肿瘤细胞生长抑制的实验研究

以反转录病毒Ｎ２Ａ为载体，使携带自身正调控序列的野生型ｐ５３基因反向插入Ｎ２Ａ两端ＬＴＲ间的Ｘｈｏｌ位点，获得了野生型ｐ５３基因反转录病毒重组体，建立了产病毒的ＰＡ３１７细胞系。用该细胞系产生的新鲜病毒颗粒感染有ｐ５３基因异常的人喉癌细胞系（Ｈｅｐ２）和纤维肉瘤细胞系（ＭＤＡＨ０４１），Ｓｏｕｔｈｅｒｎ－ｂｌｏｔ分析证实野生型ｐ５３基因反转录病毒前病毒能稳定整合在受体细胞染色体基因组上并有表达。感     

HIV-1核心蛋白Gag与IL-6共表达重组鸡痘病毒的构建及其免疫原性研究

分别将IL-6和HIV-1gag基因插入到鸡痘病毒表达载体质粒pUTAL串联启动子和复合启动子(ATI-P7．5)下游，构建了重组鸡痘病毒表达质粒pUTA-GAG-IL6。经同源重组和Western blot检测，获得重组鸡痘病毒，并将该重组病毒免疫BALB／c小鼠，检测免疫小鼠血清抗体水平和特异性CTL杀伤活性。结果获得的重组鸡痘病毒可同时表达HIV-1核心蛋白和IL-6，并在感染细胞内形成病毒样粒子。重组病毒具有良好的免疫原性，可诱导小鼠产生特异性体液免疫和细胞免疫。     

CSF－1R激酶突变子的构建及其突变体在真核细胞的表达

通过ＰＣＲ二步法，构建了ｍＣＳＦ－１Ｒ激酶负性突变子，以及野生型和突变型ＣＳＦ－１Ｒ的送病毒表达载体ｐＣＥＮ／ＭＰＳＶ。进行了１２５Ｉ－ＣＳＦ－１受体结合分析：检测了激酶负性突变子，删除了ＣＳＦ－１ＲＣ－末端氨基酸９２５以后部分的突变体（ＣＴＲＵＮＣ９２５）和删除了激酶插入区的突变体（△ＫＩ）在３２Ｄ髓细胞表达。表达水平可达１～２×１０４受体／细胞。初步测定了通过ＣＳＦ－１Ｒ所介导的促细胞分裂效应。     

猪瘟病毒E2蛋白4重复抗原表位的构建及抗原活性研究

利用PCR方法获得4次重复的猪瘟病毒E2蛋白中和性抗原表位基因，将其克隆到pGEM-5ZF( )载体，测序正确后亚克隆到pGEX-3X载体构建得到重组质粒pGEX-3X-4P。重组质粒在大肠杆菌中诱导表达了含4重复抗原表位的融合蛋白。该蛋白经纯化后，利用间接ELISA检测其与血清的反应性，结果表明，纯化的融合蛋白与兔抗CSFV E2血清有很强的反应性，与兔抗BVDV E2血清不反应，说明该重复抗原表位在鉴别诊断CSFV与猪的BVDV感染方面具有潜在的应用价值。     

动物乳腺组织高效表达通用型YAC载体的构建

以含有人α-乳白蛋白基因功能域的YAC(yeast artificial chromosome)为起始材料,构建了可以在动物乳腺高效表达外源基因的载体系统。根据已发表的人α-乳白蛋白基因序列,通过PCR的方法,扩增出了其5’端的837bp及3’端的1007bp片段,构建了整合型载体pRLA22。利用pRLA22在大肠杆菌中进行基因操作,可以将外源基因准确地置于乳白蛋白基因启动子控制之下。把重组的pRLA22导入含YAC的酵母菌后,高频率同源重组事件将会把外源基因插入YAC。本实验通过上述程序,将鸡的γ-干扰素基因cDNA序列导入了YAC。经过PCR检测,证明鸡的γ-干扰素cDNA序列已被重组到YAC的预定位置。因此,大肠杆菌载体pRLA22和含人的α-乳白蛋白基因的YAC,将构成能在动物乳腺高表达外源基因的有效载体系统。     

DNA vaccine against Taenia solium cysticercosis expressed as a modified hepatitis B virus core particle containing three epitopes shared by Taenia crassiceps and Taenia solium

Many studies have provided evidence that the hepatitis B core antigen particle is useful as a vaccine carrier for foreign epitopes. Epitopes KETc1, KETc12, and GK-1 are three promising candidates for designing a vaccine against Taenia solium cysticercosis. In the present study, epitopes KETc1 and KETc12 were inserted into the immunodominant loop of the truncated HBc149, and epitope GK-1 was fused to its C-terminus. The fused protein deltaC-3n was expressed and purified successfully. The polymeric character was tested by SDS-PAGE. After inoculation of BALB/c mice with deltaC-3n, antibody titers were assayed by ELISA, and the antibody specification was analyzed by Western blot. Dot ELISA was performed to verify the protection of the three epitopes. Results showed that the purified polymeric protein was formed, high antibody titers were induced in immunized mice and three antibodies different in molecular weight were induced, serum specific antibody recognized the native peptide localized mainly in cyst wall cells, and there was no specific antibody toward the three epitopes in sera of infected pig and humans. All these revealed that the protein deltaC-3n was a potential candidate for vaccine against cysticercosis. So the deltaC-3n sequence and the signal peptide sequence of IL-2 were cloned to a vector pVAX3.0 to construct pVAX-S-deltaC-3n. Pigs were immunized with pVAX-S-deltaC-3n. Two weeks after the immunization booster, pigs were introduced to infectious T. solium eggs. The relative protective rate induced in pigs immunized with the DNA vaccine pVAX-S-deltaC-3n was 83%.     

碳纳米管和壳聚糖的层层静电自组装多层膜(英文)

将多壁碳纳米管(MWCNT)置于混酸(硝酸∶硫酸=1∶3)中,利用超声波振荡截短碳纳米管、并使其与羧基链接,而后基于阳离子聚合电解质壳聚糖(CS)和阴离子短切碳纳米管之间的静电作用,在玻璃衬底上通过层层的模式均匀稳定地自组装形成复合壳聚糖多层膜。UV-vis光谱显示:组装过程呈现均匀而连续的生长。AFM和SEM观察表明:CS/MWCNT多层膜具有良好的光学特性,在生物传感器方面具有潜在的应用前景。     

Surface plasmon resonance shows that type IV pili are important in surface attachment by Pseudomonas aeruginosa.

Type IV pili have been shown to play a role in the early stages of bacterial biofilm formation, but not in initial bacterial attachment. Here, using the surface analytical technique, surface plasmon resonance (SPR), we follow the attachment of the bacterium Pseudomonas aeruginosa in real time. In contrast to previous studies, we show that type IV pili mutants are defective in attachment. Both mutants lacking pili (pilA), and those possessing an overabundance of pili (pilT), showed reduced SPR measured attachment compared with the wild-type PAO1 strain. Both pil mutants also showed reduced pathogenicity in a model insect host, as measured by percentage mortality after 24h. SPR revealed differences in the kinetics of attachment between pilA and pilT, differences obscured by endpoint assays using crystal violet stain. These results highlight the power of SPR in monitoring bacterial attachment in real time and also demonstrate an additional role for type IV pili beyond bacterial aggregation and micro-colony formation.     

以恒河猴为模型的恶性疟原虫DNA疫苗的免疫保护作用研究

研究了以霍乱毒素Ｂ亚基为载体的重组疟疾多价抗原的ＤＮＡ疫苗在恒河猴中的免疫原性及对相应疟原虫感染的免疫保护作用。结果表明：ＤＮＡ疫苗免疫恒河猴后,用１．２５＊１０＾８个食蟹疟子孢子攻击,对照绷带人动物在攻击后１４天左右全部感染,感染持续３４天以上;ＤＡＮ疫苗组的５只动物一直到攻击后６０天,没有感染。另外,还检测了免疫后不同时间各组的免疫应答水平,对照组相比,ＤＮＡ疫苗组免疫２次后即产生了较高水平的     

Nicotine–magnesium aluminum silicate microparticle surface modified with chitosan for mucosal delivery

Magnesium aluminum silicate (MAS), a negatively charged clay, and nicotine (NCT), a basic drug, can interact electrostatically to form microparticles. Chitosan (CS) was used for the surface modification of the microparticles, and a lyophilization method was used to preserve the original particle morphology. The microparticles were characterized in terms of their physicochemical properties, NCT content, mucoadhesive properties, and release and permeation across porcine esophageal mucosa. The results showed that the microparticles formed via electrostatic interaction between MAS and protonated NCT had an irregular shape and that their NCT content increased with increasing NCT ratios in the microparticle preparation solution. High molecular weight CS (800 kDa) adsorbed to the microparticle surface and induced a positive surface charge. CS molecules intercalated into the MAS silicate layers and decreased the crystallinity of the microparticles, leading to an increase in the release rate and diffusion coefficient of NCT from the microparticles. Moreover, the microparticle surface modified with CS was found to have higher NCT permeation fluxes and mucoadhesive properties, which indicated the significant role of CS for NCT mucosal delivery. However, the enhancement of NCT permeation and of mucoadhesive properties depended on the molecular weight and concentration of CS. These findings suggest that NCT-MAS microparticle surface modified with CS represents a promising mucosal delivery system for NCT.     

恶性疟原虫FCC1／HN株P190抗原保守区基因的克隆及其在大肠杆菌中的表达

Ｐ１９０是恶性疟原虫裂殖子表面抗原，是很有希望的疟疾疫苗候选抗原。采用ＰＣＲ方法克隆了我国海南省ＦＣＣ１／ＨＮ株Ｐ１９０抗原基因的３个保守区片段，连接到ｐＵＣ１８载体上进行ＤＮＡ序列测定，然后分别与表达载体ｐＧＥＸ－２Ｔ连接，经双酶切鉴定后转化感受态ＪＭ１０９大肠杆菌进行高效融合表达。     

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.     

Recombinant glycans on an S-layer self-assembly protein: a new dimension for nanopatterned biomaterials

Crucial biological phenomena are mediated through carbohydrates that are displayed in a defined manner and interact with molecular scale precision. We lay the groundwork for the integration of recombinant carbohydrates into a "biomolecular construction kit" for the design of new biomaterials, by utilizing the self-assembly system of the crystalline cell surface (S)-layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a. SgsE is a naturally O-glycosylated protein, with intrinsic properties that allow it to function as a nanopatterned matrix for the periodic display of glycans. By using a combined carbohydrate/protein engineering approach, two types of S-layer neoglycoproteins are produced in Escherichia coli. Based on the identification of a suitable periplasmic targeting system for the SgsE self-assembly protein as a cellular prerequisite for protein glycosylation, and on engineering of one of the natural protein O-glycosylation sites into a target for N-glycosylation, the heptasaccharide from the AcrA protein of Campylobacter jejuni and the O7 polysaccharide of E. coli are co- or post-translationally transferred to the S-layer protein by the action of the oligosaccharyltransferase PglB. The degree of glycosylation of the S-layer neoglycoproteins after purification from the periplasmic fraction reaches completeness. Electron microscopy reveals that recombinant glycosylation is fully compatible with the S-layer protein self-assembly system. Tailor-made ("functional") nanopatterned, self-assembling neoglycoproteins may open up new strategies for influencing and controlling complex biological systems with potential applications in the areas of biomimetics, drug targeting, vaccine design, or diagnostics.     

藻蓝蛋白对Hela细胞CD59基因表达调控作用的研究

探讨了钝顶螺旋藻藻蓝蛋白(PC)对Hela细胞CD59基因表达的调控作用.以正常人CD59cDNA基因为模板,经PCR扩增后重组入真核表达质粒载体pALTER-MAX,然后利用阳离子脂质体(Lipfectamine-2000)将重组质粒和PcDNA共转染人子宫颈癌细胞(Hela)和对照用正常中国仓鼠卵巢细胞(Chinese hamster ovary,CHO)进行表达.用不同浓度的钝顶螺旋藻藻蓝蛋白作用于转染细胞,通过核酸分子杂交技术、免疫荧光标记法和ELISA法对细胞中CD59分子的表达进行检测.结果表明:成功构建了重组质粒pALTER-MAX-CD59,并将其导入真核细胞(Hela,CHO),经G418筛选获得了CD59分子高效表达的细胞克隆.用藻蓝蛋白作用于筛选出的转基因细胞,证实藻蓝蛋白可促进Hela细胞表面CD59蛋白的表达并抑制Hela细胞的增殖,而对于正常CHO细胞无明显作用.     

Controlled release behaviors of chitosan/α, β- glycerophosphate thermo-sensitive hydrogels

Chitosan/α, β-glycerophosphate (CS/α, β-GP) thermo-sensitive hydrogels presented flowable solution state at low temperature and semisolid hydrogel when the ambient temperature increased. In this research, different concentrations of metronidazole encapsulated, CS and α, β-GP, as well as different acid solvents, were chosen to evaluate their influences on the drug release behaviors from CS/α, β-GP hydrogels. It was found that there was a sustaining release during the first 3 h followed by a plateau. SEM images showed that drugs were located both on the surface and in the interior of hydrogels. The optimal preparation conditions of this hydrogel for drug release were as follows: 1.8% (w/v) CS in HAc solvent, 5.6% (w/v) α, β-GP and 5 g/L metronidazole encapsulation. Cytotoxicity evaluation found no toxic effect. In order to control the release rate, 2.5 g/L chitosan microspheres with spherical shape and smooth surface were incorporated, and it was found that the initial release process was alleviated, while drug concentration had no obvious effect on the release rate. It could be concluded that the metronidzole release behaviors could be optimized according to practical applications.