【摘要】：目的:擴增日本血吸蟲新基因Sj Dna J-類似蛋白基因;構(gòu)建pWR450-1/Sj Dna J-類似蛋白基因原核表達載體并進行表達;構(gòu)建pcDNA3.1(+)/Sj Dna J-類似蛋白核酸疫苗,初步研究該核酸疫苗對BALB/c小鼠誘導的免疫保護性作用。 方法:根據(jù)新基因的生物信息學分析結(jié)果,選擇Sj Dna J-類似蛋白作為研究對象,用PRIMER5.0引物設計軟件設計引物并合成,PCR擴增Sj Dna J-類似蛋白基因,將PCR產(chǎn)物純化后與pUCm-T載體連接,經(jīng)藍白篩選、雙酶切和PCR鑒定后,亞克隆入pWR450-1原核表達載體及pcDNA3.1(+)真核表達載體中,經(jīng)抗生素(氨芐)篩選、雙酶切鑒定、PCR鑒定及測序鑒定后,將構(gòu)建的pWR450.1/Sj Dna J-類似蛋白基因重組體于大腸埃希菌中以IPTG誘導表達,SDS-PAGE及Western blotting鑒定表達情況;構(gòu)建的pcDNA3.1(+)/Sj Dna J-類似蛋白基因重組質(zhì)粒經(jīng)大量擴增后,注射到BALB/c小鼠后腿股四頭肌肌肉組織中,每2周免疫一次,共免疫3次,末次免疫后2周,用日本血吸蟲尾蚴感染BALB/c免疫小鼠,42天后剖殺,計算肝臟蟲卵以及成蟲數(shù),并用PCR、免疫組化法、ELISA等方法對Sj Dna J-類似蛋白基因在小鼠體內(nèi)的表達、穩(wěn)定性以及對小鼠的保護性作用等進行研究。 結(jié)果:構(gòu)建了pWR450.1/Sj Dna J-類似蛋白基因重組原核表達載體,并在大腸埃希菌TG1中經(jīng)IPTG誘導后表達。構(gòu)建了pcDNA3.1(+)/Sj Dna J-類似蛋白基因真核表達重組體,免疫BALB/c小鼠后,提取重組質(zhì)粒組小鼠后腿股四頭肌的總DNA,PCR擴增在一定時期內(nèi)能檢測到Sj Dna J-類似蛋白基因;免疫組化實驗顯示Sj Dna J-類似蛋白基因可在免疫小鼠肌肉中表達;末次免疫14天后,重組質(zhì)粒組小鼠血
[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.
【學位授予單位】：南華大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R383

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