日本血吸蟲Dna J-類似蛋白基因原核、真核表達(dá)載體的構(gòu)建及其對小鼠的保護(hù)性免疫作用研究
發(fā)布時間：2019-03-30 17:29
【摘要】：目的:擴(kuò)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(dá)載體并進(jìn)行表達(dá);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(dǎo)的免疫保護(hù)性作用。 方法:根據(jù)新基因的生物信息學(xué)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(shè)計(jì)軟件設(shè)計(jì)引物并合成,PCR擴(kuò)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(lán)白篩選、雙酶切和PCR鑒定后,亞克隆入pWR450-1原核表達(dá)載體及pcDNA3.1(+)真核表達(dá)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(dǎo)表達(dá),SDS-PAGE及Western blotting鑒定表達(dá)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(kuò)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(jì)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(dá)、穩(wěn)定性以及對小鼠的保護(hù)性作用等進(jìn)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(dá)載體,并在大腸埃希菌TG1中經(jīng)IPTG誘導(dǎo)后表達(dá)。構(gòu)建了pcDNA3.1(+)/Sj Dna J-類似蛋白基因真核表達(dá)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(kuò)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(shí)驗(yàn)顯示Sj Dna J-類似蛋白基因可在免疫小鼠肌肉中表達(dá);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(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R383
本文編號：2450321
[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(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R383
【參考文獻(xiàn)】
相關(guān)期刊論文 前6條
1 李萍,嚴(yán)家新;DNA疫苗研究進(jìn)展[J];國外醫(yī)學(xué).預(yù)防.診斷.治療用生物制品分冊;1995年03期
2 宋磊 ,劉紅軍,郭大東;核酸疫苗——未來的疫苗[J];山東醫(yī)藥工業(yè);2002年01期
3 金亞美,林矯矯,張亮,傅志強(qiáng),吳祥甫,周元聰,蔡幼民;日本血吸蟲中國大陸株21.7kD蛋白編碼基因的克隆和表達(dá)[J];生物工程學(xué)報(bào);2002年06期
4 唐小牛,汪學(xué)龍;日本血吸蟲(大陸株)26ku谷胱甘肽轉(zhuǎn)移酶基因真核表達(dá)載體構(gòu)建及序列分析[J];皖南醫(yī)學(xué)院學(xué)報(bào);2003年01期
5 陳名剛;世界血吸蟲病流行情況及防治進(jìn)展[J];中國血吸蟲病防治雜志;2002年02期
6 李德發(fā);血吸蟲核酸疫苗研究進(jìn)展[J];中國血吸蟲病防治雜志;2002年04期
，本文編號：2450321
本文鏈接：http://sikaile.net/yixuelunwen/binglixuelunwen/2450321.html
最近更新
教材專著
