日本血吸虫Dna J-类似蛋白基因原核、真核表达载体的构建及其对小鼠的保护性免疫作用研究

发布时间：2019-03-30 17:29

【摘要】：目的:扩增日本血吸虫新基因Sj Dna J-类似蛋白基因;构建pWR450-1/Sj Dna J-类似蛋白基因原核表达载体并进行表达;构建pcDNA3.1(+)/Sj Dna J-类似蛋白核酸疫苗,初步研究该核酸疫苗对BALB/c小鼠诱导的免疫保护性作用。方法:根据新基因的生物信息学分析结果,选择Sj Dna J-类似蛋白作为研究对象,用PRIMER5.0引物设计软件设计引物并合成,PCR扩增Sj Dna J-类似蛋白基因,将PCR产物纯化后与pUCm-T载体连接,经蓝白筛选、双酶切和PCR鉴定后,亚克隆入pWR450-1原核表达载体及pcDNA3.1(+)真核表达载体中,经抗生素(氨苄)筛选、双酶切鉴定、PCR鉴定及测序鉴定后,将构建的pWR450.1/Sj Dna J-类似蛋白基因重组体于大肠埃希菌中以IPTG诱导表达,SDS-PAGE及Western blotting鉴定表达情况;构建的pcDNA3.1(+)/Sj Dna J-类似蛋白基因重组质粒经大量扩增后,注射到BALB/c小鼠后腿股四头肌肌肉组织中,每2周免疫一次,共免疫3次,末次免疫后2周,用日本血吸虫尾蚴感染BALB/c免疫小鼠,42天后剖杀,计算肝脏虫卵以及成虫数,并用PCR、免疫组化法、ELISA等方法对Sj Dna J-类似蛋白基因在小鼠体内的表达、稳定性以及对小鼠的保护性作用等进行研究。结果:构建了pWR450.1/Sj Dna J-类似蛋白基因重组原核表达载体,并在大肠埃希菌TG1中经IPTG诱导后表达。构建了pcDNA3.1(+)/Sj Dna J-类似蛋白基因真核表达重组体,免疫BALB/c小鼠后,提取重组质粒组小鼠后腿股四头肌的总DNA,PCR扩增在一定时期内能检测到Sj Dna J-类似蛋白基因;免疫组化实验显示Sj Dna J-类似蛋白基因可在免疫小鼠肌肉中表达;末次免疫14天后,重组质粒组小鼠血
[Abstract]:Objective: to amplify the Sj Dna J-like protein gene of Schistosoma japonicum, construct the prokaryotic expression vector of pWR450-1/Sj Dna J-like protein gene and express it. To construct pcDNA3.1 () / Sj Dna J-like protein nucleic acid vaccine, and to study the immune protective effect of the nucleic acid vaccine on BALB/c mice. Methods: according to the results of bioinformatics analysis of the new gene, Sj Dna J-like protein was selected as the research object, primers were designed and synthesized by PRIMER5.0 primer design software, and Sj Dna J-like protein gene was amplified by PCR, and Sj Dna J-like protein gene was amplified by PCR. The PCR product was purified and ligated with pUCm-T vector. After blue-white screening, double enzyme digestion and PCR identification, the product was subcloned into pWR450-1 prokaryotic expression vector and pcDNA3.1 () eukaryotic expression vector, screened by antibiotic (ampicin) and identified by double enzyme digestion. After PCR identification and sequencing identification, the recombinant pWR450.1/Sj Dna J-like protein gene was induced by IPTG in Escherichia coli and identified by SDS-PAGE and Western blotting. The recombinant plasmid of pcDNA3.1 () / Sj Dna J-like protein gene was amplified and injected into the muscle tissue of quadriceps femoris muscle of BALB/c mice. The recombinant plasmid was immunized every 2 weeks, 3 times, and 2 weeks after the last immunization, the recombinant plasmid was injected into the muscle tissue of the quadriceps femoris muscle of the hind leg of BALB/c mice. Mice were immunized with cercariae of Schistosoma japonicum (BALB/c) and killed 42 days later to calculate the number of liver eggs and adults. The expression of Sj Dna J-like protein gene in mice was detected by PCR, immunohistochemistry and ELISA. The stability and protective effect on mice were studied. Results: the recombinant prokaryotic expression vector of pWR450.1/Sj Dna J-like protein gene was constructed and expressed in Escherichia coli TG1 induced by IPTG. The eukaryotic expression recombinant of pcDNA3.1 () / Sj Dna J-like protein gene was constructed. After immunizing BALB/c mice, the total DNA, of quadriceps femoris muscle of the hind leg was extracted from the recombinant plasmid group. Sj Dna J-like protein gene could be detected by PCR amplification in a certain period of time. Immunohistochemical analysis showed that Sj Dna J-like protein gene could be expressed in the muscle of immunized mice. 14 days after the last immunization, the blood of mice in the recombinant plasmid group was treated with the recombinant plasmid.
【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】：R383

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