在杂合启动子控制下乙肝病毒表面抗原SA-28融合基因在酵母中的表达及调控

采用酵母杂合启动子PADH2－CUP1或PADH2－GAPDH及终止子TADH1，构建了一系列酵母表达载体．在这些表达载体中插入乙肝病毒表面抗原S－preS1融合基因SA－28后，将合乙肝病毒表面抗原的表达单元克隆至高稳定质粒PHC11的BamHI位点．然后将表达质粒分别转化酿酒酵母Y16，Y19．对SA－28基因表达的研究表明，在酵母菌胞内实现了SA－28基因的高表达，且表达受葡萄糖浓度调控．     

HIV-1和HBV复合型DNA免疫的初步研究

近年的研究表明,在啮齿类和非人灵长类免疫带有编码病毒和细菌抗原基因的质粒ＤＮＡ可以激发体液和细胞免疫应答．在本实验中,将ＨＢＶ的Ｓ基因和ＨＩＶ－１的ｇｐ１６０基因以融合形式插入到载体ｐｃＤＮＡ３中,其能表达ＨＢｓＡｇ和ｇｐ１６０的融合蛋白,并将此质粒ＤＮＡ分别直接注射到Ｂａｌｂ／ｃ小鼠和Ｓｗｉｓ小鼠．三次免疫后,用ＥＬＩＳＡ的方法初步检测ＨＢｓＡｇ和ｇｐ１６０抗原特异的抗体免疫应答均为阳性．结果说明,带有ＨＢＶ和ＨＩＶ－１融合基因的质粒ＤＮＡ直接免疫小鼠后,均激发了小鼠产生相应的免疫应答反应,这个结果为研究和生产多价疫苗提供了新的思路     

水动力转染技术建造慢性乙型肝炎动物模型特效研究

摘要: 目的比较不同质粒建立的水动力转染乙型肝炎小鼠模型的特性。方法将pcDNA 3. 1-1. 3-HBV-C 与PAAV-1. 2-HBV-A 两种质粒通过水动力转染法建立两种乙型肝炎小鼠模型,通过对建模成功率和小鼠外周血HBsAg 滴度及稳定性,T 细胞指数流式等检测分析,比较不同模型差异和特效。结果转染pcDNA3. 1-1. 3-HBV-C质粒建模成功率为78. 6%,转染后体内HBsAg 滴度最高达到220IU/mL; 转染PAAV-1. 2-HBV-A 质粒建模成功率为56%,转染后体内HBsAg 滴度最高达到2 300IU/mL。建模小鼠的肝脏进行切片,做免疫组化后,观察实验结果,转染pcDNA 3. 1-1. 3-HBV-C 质粒的小鼠肝脏切片的HBsAg 免疫组化较PAAV-1. 2-HBV-A 质粒小鼠颜色面积更大。实验小鼠转染质粒后6 周的外周血进行流式细胞术检测,结果显示特异性T 细胞CD4 +、CD8 + 和正常小鼠比较,两种质粒的造模小鼠未有差别。结论利用水动力转染法用不同质粒建造乙型肝炎小鼠模型在乙肝病毒指标水平和稳定性方面有着显著差异。     

Enhancing cellular immune response to HBV M DNA vaccine in mice by codelivery of interleukin-18 recombinant

OBJECTIVE: To investigate the effect of interleukin-18 (IL-18) on immune response induced by plasmid encoding hepatitis B virus middle protein antigen and to explore new strategies for prophylactic and therapeutic HBV DNA vaccines. METHODS: BALB/c mice were immunized with pCMV-M alone or co-immunized with pcDNA3-18 and pCMV-M and then their sera were collected for analysing anti-HBsAg antibody by ELISA; splenocytes were isolated for detecting specific CTL response and cytokine assay in vitro. RESULTS: The anti-HBs antibody level of mice co-immunized with pcDNA3-18 and pCMV-M was slightly higher than that of mice immunized with pCMV-M alone, but there was not significantly different (P>0.05). Compared with mice injected with pCMV-M, the specific CTL cytotoxity activity of mice immunized with pcDNA3-18 and pCMV-M was significantly enhanced (P<0.05) and the level of IFN-Gamma in supernatant of splenocytes cul-tured with HBsAg in vitro was significantly elevated (P<0.05) while the level of IL-4 had no significant difference (P>0.05). CONCLUSION: The plasmid encoding IL-18 together with HBV M gene DNA vaccines may enhance specific TH1 cells and CTL cellular immune response induced in mice, so that IL-18 is a promising immune adjuvant.     

顶头孢霉cefG基因的表达及抗体制备的研究

通过PCR方法得到带酶切位点的cefG基因,用于构建表达质粒pET30-G,并在大肠杆菌里成功表达出乙酰转移酶的包涵体。将该重组蛋白的标准品作为抗原,免疫BALB/c小鼠得到多克隆抗体,酶联免疫吸附测定(ELISA)分析得抗体的效价满足实验需求。利用该抗体绘制出抗原浓度的标准曲线,确定待测抗原适合的检测浓度在0.16μg/mL以下。制得的抗体在重组大肠杆菌里检测到了乙酰转移酶,证明所得抗体具有实验价值。     

乙肝病毒表面抗原基因植物表达载体的构建

报道了将乙型肝炎病毒（ａｄｗ亚型）表面抗原（ＨＢｓＡｇ）基因和ＨＢＡｇ及其前导序列（ＨＢｓＡｇ＋ｐｒｅＳ２）基因分别插入植物表达载体ｐＲｏＫⅡ的ＣａＭＶ３５Ｓ启动子下游,构建为重组质粒ｐＲＨＢ和ｐＲＰ,并将其分别导入农杆菌ＬＢＡ４４０４中。     

以减毒猪霍乱沙门菌为载体的抗O型口蹄疫病毒重组活菌苗在家兔体内免疫应答的初步研究

将含有口蹄疫病毒疫苗基因的真核表达质粒pBO1经过中间宿主鼠伤寒沙门菌LB5010修饰后,电转化到减毒的猪霍乱沙门菌C500中,构建成抗O型口蹄疫病毒的重组活菌苗pBO1／S．cho．血清凝集实验验证,筛选到的转化菌仍然具有猪霍乱沙门菌的血清学特性,即具有免疫原性．采用口服方式免疫家兔,检测重组菌在家兔体内诱导的免疫应答．家兔免疫后8周,分离脾脏T细胞,检测T细胞增殖情况．结果显示,口服DBO1／S．fho的家兔T细胞在FMDV特异性抗原的刺激下有明显的增殖反应,刺激指数SI〉11;免疫后2周和8周分别取家兔静脉血,用液相阻断ELISA法检测血清中针对FMDV的特异性抗体,pBO1／S．cho组家兔产生的抗体效价为1／32。     

CpG ODN增强rHBsAg免疫小鼠抗体产生及淋巴细胞增殖效应

目的：探讨合成含CpG基序的寡核苷酸（CpG ODN）对重组乙型肝炎表面抗原（rHBsAg ）增强小鼠特异性抗体产生及淋巴细胞增殖反应的效应.方法：采用BaIb/c小鼠作为免疫实验动物,经后腿胫骨前肌免疫2次,ELISA法检测血清乙型肝炎表面抗体（抗-HBs）效价;3H-TdR掺入法测定淋巴细胞增殖反应.结果：加CpG ODN组的抗-HBs效价较rHBsAg组明显增高,且维持时间长;淋巴细胞增殖反应显著增强.结论：CpG ODN对小鼠抗-HBs产生具有明显的增强作用,且可以有效刺激小鼠淋巴细胞的增殖反应.     

CpG DNA enhances the immune responses elicited by the DNA vaccine against foot-and-mouth disease virus in guinea pigs

CpG DNA is DNA sequence that has immune stimulatory effects. Several lines of investigation over the past few years indicate that CpG DNA plays an important role in the induction of immune responses to DNA vaccines. In this study, CpG DNA-containing synthetic oligodeoxynucleotide (CpG-ODN) was cloned into the eukaryotic expression plasmid encoding a fusion protein containing b- galactosidase from E. coli and immunogenic epitopes of foot- and-mouth disease virus (FMDV) type O, and the immune responses induced by the plasmid were assayed. The results showed that guinea pigs immunized with the recombinant plasmid containing CpG-ODN generated a higher level of FMDV-neutralizing antibody and a stronger T cell proliferative response and protection against viral challenge than those receiving the plasmid containing no CpG-ODN. Our study demonstrated that it is an effective route to enhance the efficacy of DNA vaccines by inserting exogenous CpG DNA into the plasmids, and the DNA vaccine developed here is a promising candidate to prevent FMDV infection.     

肝片吸虫DNA疫苗的构建及其免疫效应的初步研究

用ＥcorRI酶切含肝片吸虫保护性抗原基因ＦＨ３的重组质粒pUC18/FH3,回收ＦＨ３（－１．０Ｋb)片段克隆到真核表达载体pBlueCMV上,酶切筛选顺向插入重组子pBlueCMV-FH3,即得到一种肝片吸虫ＤＮＡ疫苗,制备纯化该疫苗并免疫小鼠,小鼠肌肉细胞中有一定的ＦＨ３抗原表达;用ＥＬＩＳＡ检测抗血清表明,注射疫苗的小鼠均伴随产生一定量的特异性抗体,用肝片吸早囊蚴攻击（２０囊蚴／鼠）小鼠,初步显示有一定的减虫率。     

转乙肝病毒表面抗原基因烟草发根的获得

构建了乙肝病毒表面抗原基因（HBsAg）植物表达载体,通过冻融法将HBsAg 基因转入到发根农杆菌LBA1314中,采用叶盘法将HBsAg基因导入到烟草中,获得了转基因烟草发根.对转基因烟草发根的GUS检测结果表明：转HBsAg基因烟草发根可以染成蓝色,而非转基因烟草的根没有染色反应.这说明gus基因在转HBsAg基因烟草发根获得了表达.     

恶性疟原虫复合多表位基因的原核表达和多克隆抗体制备

原核表达恶性疟原虫多期多表位基因并制备其多克隆抗体。在前期构建恶性疟原虫复合多期多表位重组真核表达载体的基础上,将测序正确的AMAMEG基因克隆入原核表达载体pGEX4T—1并诱导表达。采用包涵体洗涤的方式纯化目的蛋白,以AMA—1抗体对纯化蛋白进行Western—blot分析。将纯化的融合蛋白免疫小鼠制备多克隆抗体。通过PCR成功获得长度为1 000 bp的恶性疟原虫AMA—1胞外域基因片段,获得了与多表位基因连接后的AMAMEG基因。通过诱导表达,显示在相对分子量约95 000处有预计大小的特异性条带,表明成功表达出融合蛋白。经此纯化的融合蛋白免疫的小鼠能产生特异性抗体,其滴度达到1∶105。纯化的融合蛋白和多克隆抗体为研究新型疟疾疫苗奠定一定的理论和实践基础。     

可降解淀粉酿酒酵母基因工程菌的构建及其生产应用

将酿酒酵母的rDNA片段,黑曲霉葡萄糖淀粉酶基因表达盒及G418抗性基因表达盒重组进经过改造的质粒pSP72,构建酿酒酵母整合型质粒YIp4RGAn及YIp19RGAn,转化酿酒酵母实验室菌株GRF18、生产菌株JL108、SD和JM,获得能高效表达葡萄糖淀粉酶和分解淀粉的酿酒酵母基因工菌。Southern印迹分析证明,葡萄糖淀粉酶基因已整合进工程菌染色体。这些工程菌在含有20%淀粉的培养基中培养,产酒率都在11%以上。     

Construction of multivalent DNA vaccines forMycobacterium tuberculosis and its immunogenicity

The coding regions of Ag85B MPT-64, and ESAT-6 secreted proteins were cloned initially into the eukaryotic expression vector pJW4303, then transformed to E. coli Top 10 strain for plasmid DNA extraction and further analysis. Plasmids containing the right insertion were sequenced to confirm their identity. COS7 cells were transfected with a mixture containing serially diluted plasmid DNA encoding three secreted proteins and Lipofectin (Gibco). The supernatants and pellets prepared from various cell lines were run on SDS-PAGE gel and the expression of these proteins in COS7 cells were demonstrated by immunoblot using polyclonal or monoclonal antiserum of M.TBH37Rv. 21 days after first vaccination of C57BL-6 mice by all three recombinant eukaryotic expressing vectors, antibody titer for Ag85B reached 1∶3200. 21 days after second vaccination, the antibody titer reached 1∶102400. The highest antibody levels induced by multivalent vaccines after the second injection were equal to or even greater than the highest antibody levels of single DNA vaccine reported in literature after third injections. Antibody titer of MPT-64 was 1∶50 after the first injection and it reached 1∶200 after the second injection. No antigen-specific antibody against ESAT-6 was detected in sera harvested from immunized mice 21 days after both injections. Antigen-specific IFN-g level of Ag85B was 110 pg/mL while no antigen-specific IFN- g level of ESAT-6 and MPT-64 was detected even after third injections. To our knowledge, it is the first time that studies of polyvalent recombinant DNA vaccines against TB were carried out in C57BL-6 mice. Our results indicated that multiple DNA vaccines could be used to enhance protective responses against M.TB.     

长爪沙鼠CST3序列同源性分析及蛋白体外表达

目的 分析长爪沙鼠半胱氨酸蛋白酶抑制剂C (Cystatin C,CST3)cDNA序列同源性,并建立CST3蛋白原核表达体系,为长爪沙鼠CST3抗体制备和后续基因功能研究奠定基础。方法 对长爪沙鼠Cst3 cDNA序列进行克隆、同源性分析及密码子优化;将优化后序列酶切连接到pET28a载体,完成重组CST3蛋白表达载体构建;将该载体转化到感受态细胞中,通过异丙基硫代半乳糖苷(Isopropyl β-D-Thiogalactoside,IPTG)诱导实现CST3蛋白的原核表达,并用SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹法(Western blotting)验证。结果 长爪沙鼠Cst3基因与人和小鼠Cst3基因的序列同源性较低。密码子优化后的长爪沙鼠Cst3表达序列插入到pET28a质粒中,获得了CST3蛋白表达载体。经1 mmol/L IPTG 37 ℃诱导12 h,可获得大量CST3蛋白。结论 成功地构建了长爪沙鼠CST3蛋白的体外表达体系。     

BALB／c小鼠选择与腹水产量和抗体效价间关系的研究

目的观察在鼠疫F1单克隆抗体制备过程中,BALB／c小鼠的选择和饲养与所获腹水量和抗体效价的关系,为大量制备该单克隆抗体提供实验室参考依据。方法根据小鼠周龄、性别和饲养条件对BALB／c小鼠进行分组,并采用动物体内诱生法制备腹水,间接ELISA用来检测腹水中F1单克隆抗体效价。结果12～16周龄组、雄性和加强营养组小鼠所获腹水产量和效价均高于其它周龄组和对照组。结论选择适当周龄的雄性小鼠、饲养过程中加强营养和管理可适当提高腹水产量和抗体的效价。     

IL-2与猪瘟病毒E_2基因真核双表达载体的构建及其免疫增强作用的研究

应用DNA重组技术构建了猪瘟病毒E2 基因与IL 2基因的双表达真核表达载体 ,观察其表达水平 ,并对其免疫增强效果进行了观察。结果是构建了猪瘟病毒E2 基因与白细胞介素 2的真核表达质粒pIRSTIL 2。将质粒转染BHK 2 1细胞 ,可在体外表达E2 和有生物活性IL 2。pIRSTIL 2质粒能诱导产生CSFV的特异性免疫反应。pIRSTIL 2所诱导的免疫应答反应比使用单表达质粒pIRST强。实验表明 :IL 2与猪瘟病毒E2 基因构建的双表达基因疫苗能有效提高基因疫苗的免疫效果     

HBV包膜大蛋白、中蛋白和HBsAg疫苗免疫原性的比较

目的:检测HBV包膜大蛋白(L蛋白)、中蛋白(M蛋白)和HBsAg疫苗的免疫原性.方法:采用本实验室表达纯化得到的HBV包膜大蛋白、中蛋白与市售疫苗(主要成分为HBsAg),以2、10g剂量免疫家兔.ELISA检测抗HBsAg抗体滴度水平变化.结果:注射了L蛋白、M蛋白和HBsAg疫苗的家兔实验组产生的抗HBsAg抗体滴度水平有着非常明显的差别.注射了L蛋白、M蛋白的家兔在14d即可检测出抗HBsAg抗体,而注射HBsAg的家兔分别在21d和28d才能检测到抗HBsAg抗体.同时注射L蛋白实验组产生的抗HBsAg抗体滴度水平要比注射同剂量HBsAg实验组高出50%,并且注射2gL蛋白的实验组比注射10gHBsAg蛋白的实验组更快出现了抗HBsAg抗体,且抗体滴度水平更高.结论:L蛋白比M蛋白及市售疫苗的免疫原性强,不但产生抗体时间提前,而且滴度高,是新一代高效乙肝疫苗的优选蛋白.     

High-frequency transformation of the fission yeast Schizosaccharomyces pombe

The fission yeast, Schizosaccharomyces pombe, has been used extensively for genetic studies but until now it has not been utilized as a host organism for DNA cloning. Here we describe a method for high-frequency transformation fo a leu 1(-) strain of this yeast with hybrid plasmids containing the Saccharomyces cerevisiae LEu 2(+) gene, a bacterial plasmid and either the S. cerevisiae 2 μm plasmid or autonomously replicating sequences (ars)(1) derived from S. pombe DNA. Some of the plasmids contain unique restriction sites which make them suitable for the isolation of S. pombe genes, and they can also be used for the exchange of DNA between S. pombe and S. cerevisiae.     

腺病毒介导ERK-2基因在生长板软骨细胞中的表达

目的:构建ERK-2基因重组腺病毒载体,检测构建的腺病毒感染原代大鼠生长板软骨细胞的效率以及目的基因的表达。方法:将ERK-2 cDNA亚克隆到腺病毒穿梭载体pAdTrack-CMV中,线性化后与腺病毒骨架质粒pAdEasy-1共同转染E.Col.i B J5183,将筛选、鉴定的重组腺病毒质粒线性化后转染HEK293细胞进行病毒颗粒的包装;流式细胞术检测不同感染复数(MO I)ERK-2重组腺病毒感染原代培养的大鼠肋生长板软骨细胞的效率,W estern b lot检测腺病毒感染的生长板软骨细胞中ERK-2蛋白的表达。结果:成功构建ERK-2重组腺病毒,MO I 50的腺病毒感染原代生长板软骨细胞的效率大于90%,感染的生长板软骨细胞中ERK-2表达显著增加。结论:构建的重组腺病毒可介导ERK-2基因在原代大鼠生长板软骨细胞中高表达。