SARS相关冠状病毒S1基因片段真核表达载体的构建及其免疫活性测定

发布时间：2018-01-02 22:44

本文关键词：SARS相关冠状病毒S1基因片段真核表达载体的构建及其免疫活性测定　出处：《南华大学》2005年硕士论文　论文类型：学位论文

【摘要】：目的:根据GeneBank登录的BJ01株SARS-CoV核酸序列合成801bp S1基因片段(22382bp～23182bp),构建SARS-CoV相关冠状病毒S1基因片段真核表达载体,并肌注免疫BALB/c小鼠,观察小鼠所产生的体液免疫和细胞免疫应答水平,从而为SARS-CoV生物学活性的研究及核酸疫苗的研制提供实验依据。方法:用PRIMER5.0引物设计软件设计引物,聚合酶链反应(PCR)扩增S1基因片段801bp。将PCR产物纯化后与pUCm-T载体连接,经蓝白斑筛选、双酶切鉴定及序列分析后,亚克隆至pcDNA3.1(+)真核表达载体中;阳性克隆经双酶切鉴定后转染HeLa细胞,免疫细胞化学法与Western-Blotting鉴定其在真核细胞中的表达;以pcDNA3.1(+)/S1肌注免疫6周龄BALB/C小鼠,PCR与免疫组化法检测SARS相关冠状病毒S1基因在小鼠股四头肌的表达,ELISA法检测抗SARS-CoV IgG,MTT法检测免疫小鼠的T细胞增殖活性,并检测免疫小鼠脾淋巴细胞的IFN-γ水平。结果:扩增的SARS-CoVS1基因片段801bp亚克隆至pcDNA3.1(+)/S1后,免疫细胞化学试验与Western-Blotting结果表明pcDNA3.1(+)/S1可以在HeLa细胞中表达大小约为32KD的S1蛋白。免疫组织化学结果显示pcDNA3.1(+)/S1免疫组小鼠股四头肌细胞内表达了SARS-CoV S1蛋白。免疫后小鼠的体液与细胞免疫应答水平均随免疫时间增强。随着免疫次数的增加和接种时间的延续,pcDNA3.1(+)/S1免疫组小鼠体内抗SARS-CoV IgG量明显升高,抗体滴度达1∶2000。T淋巴细胞增殖实验表明,pcDNA3.1(+)/S1注射组细胞加入刺激物后,细胞成团生长,pET-22b/S1重组蛋白刺激组较PHA刺激组细胞增殖明显活跃,各组间有显著性差异(采用方差分析,F=12.045,P=0.000,P0.05);细胞因子IFN-γ检测
[Abstract]:Objective: to synthesize 801bp S1 gene fragment (22382bphp) according to the nucleic acid sequence of BJ01 strain SARS-CoV entered by GeneBank. The eukaryotic expression vector of S1 gene fragment of SARS-CoV associated coronavirus was constructed and BALB/c mice were immunized intramuscularly to observe the level of humoral immunity and cellular immune response. It provides experimental basis for the study of SARS-CoV biological activity and the development of nucleic acid vaccine. Methods: the primers were designed with PRIMER5.0 primer design software. The S1 gene fragment was amplified by polymerase chain reaction (PCR). The PCR product was purified and ligated with pUCm-T vector. Subcloned into eukaryotic expression vector pcDNA3.1 (); The positive clones were identified by double enzyme digestion and transfected into HeLa cells. Their expression in eukaryotic cells was identified by immunocytochemistry and Western-Blotting. The expression of SARS associated coronavirus S1 gene in the quadriceps femoris of BALB/C mice at 6 weeks of age was detected by pcDNA3.1 (intramuscular injection) and immunohistochemical method. The activity of T cell proliferation and the level of IFN- 纬 in spleen lymphocytes of immunized mice were detected by ELISA assay. Results: the amplified SARS-CoVS1 gene fragment 801bp was subcloned into pcDNA3.1 (P / S1). The results of immunocytochemistry and Western-Blotting showed that pcDNA3.1 (). RS1 could express about 32KD S1 protein in HeLa cells. Immunohistochemical results showed that pcDNA3.1 (). SARS-CoV was expressed in quadriceps femoris cells of mice immunized with rS1. S1 protein. The humoral and cellular immune response levels of mice after immunization increased with the immune time, with the increase of immunization times and the extension of inoculation time. The antibody titer was 1: 2000.T lymphocyte proliferation test showed that the amount of anti SARS-CoV IgG was significantly increased in mice immunized with pcDNA3.1 (P / S 1). After the cells were injected with pcDNA3.1, the proliferation of pET-22bS1 recombinant protein stimulated by pET-22bS1 recombinant protein was more active than that of PHA group. There was significant difference among the three groups (using the analysis of variance analysis of variance (AANOVA) 12.045A, P0. 000P0. 05); Cytokine IFN- 纬 detection
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R373

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