本文關(guān)鍵詞： 日本血吸蟲 組織蛋白酶B 基因克隆 原核表達(dá)　出處：《福建醫(yī)科大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的:構(gòu)建表達(dá)日本血吸蟲(Schistosoma japonicum)組織蛋白酶B(Cathepsin B,CB/ Sj31)基因的原核表達(dá)系統(tǒng),優(yōu)化表達(dá)條件,以期獲得重組組織蛋白酶B的高效表達(dá),為進(jìn)一步研究組織蛋白酶B的生化特性和生物學(xué)功能奠定基礎(chǔ)。 方法: 1.基因克隆:提取日本血吸蟲總RNA。Genebank中獲得Sj31基因全長(zhǎng),通過RT-PCR擴(kuò)增Sj31基因的cDNA序列(含768個(gè)堿基),基因克隆技術(shù)構(gòu)建pET30a- Sj31重組表達(dá)載體。采用氯化鈣共沉淀法將重組子轉(zhuǎn)化至E.coli DH5α和BL21(DE3)。PCR、酶切、測(cè)序方法篩選陽(yáng)性重組子。 2.蛋白表達(dá):①抗血清的制備:取感染日本血吸蟲的大白兔心臟血,分離取血清。經(jīng)大腸桿菌裂解液吸附法處理。②IPTG誘導(dǎo)BL21(DE3)細(xì)菌重組Sj31蛋白表達(dá),經(jīng)SDS-PAGE電泳及用5×His單抗(小鼠源)抗原鑒定融合蛋白。用兔抗日本血吸蟲多克隆抗體,檢測(cè)蛋白的抗原性。優(yōu)化誘導(dǎo)表達(dá)的時(shí)間和IPTG的誘導(dǎo)濃度,誘導(dǎo)重組蛋白大量表達(dá)。 結(jié)果: 1.提取了日本血吸蟲成蟲的總RNA。兩步法RT-PCR擴(kuò)增出Sj31基因的cDNA序列�；蚩寺〖夹g(shù)成功構(gòu)建pET30a- Sj31重組表達(dá)載體。 2.重組子可被IPTG誘導(dǎo)表達(dá),誘導(dǎo)表達(dá)的重組蛋白可被5×His單抗(小鼠源)抗原識(shí)別;并且重組蛋白可被兔抗日本血吸蟲多克隆抗體識(shí)別。當(dāng)誘導(dǎo)表達(dá)時(shí)間為4h、IPTG終濃度為1.0mmol/L時(shí),表達(dá)量最大。 結(jié)論:成功構(gòu)建Sj31基因的原核表達(dá)系統(tǒng),獲得高效表達(dá);重組蛋白肽段具有良好的抗原性。為進(jìn)一步研究其功能打下了必要的基礎(chǔ)。
[Abstract]:Objective: to construct a cathepsin B (Cathepsin B) expressing Schistosoma japonicum (Schistosoma japonicum) from Schistosoma japonicum. The prokaryotic expression system of CBSj31) gene was optimized to obtain the high expression of recombinant cathepsin B. It will lay a foundation for the further study of the biochemical characteristics and biological functions of cathepsin B. Methods: 1. Gene cloning: the full length of Sj31 gene was obtained from total RNA.Genebank of Schistosoma japonicum. The cDNA sequence of Sj31 gene (including 768 bases) was amplified by RT-PCR. The recombinant expression vector pET30a- Sj31 was constructed by gene cloning. The recombinant plasmid was transformed into E. coli DH5 偽 and BL21(DE3).PCR by calcium chloride coprecipitation. The positive recombinant was screened by enzyme digestion and sequencing. 2. Preparation of antiserum against protein expression of 1: 1: blood from heart of rabbits infected with Schistosoma japonicum. The recombinant Sj31 protein expression of BL21DE3 was induced by E. coli lytic solution adsorption. The fusion protein was identified by SDS-PAGE electrophoresis and 5 脳 His monoclonal antibody (mouse origin). Rabbit polyclonal antibody against Schistosoma japonicum was used. To determine the antigenicity of the protein, to optimize the time of induction and the concentration of IPTG, and to induce the expression of recombinant protein in large quantities. Results: 1. The total RNA of adult Schistosoma japonicum was extracted. The cDNA sequence of Sj31 gene was amplified by two-step RT-PCR. The gene cloning technique successfully constructed pET30a-. Sj31 recombinant expression vector. 2.Recombinant can be induced to express by IPTG, and the recombinant protein can be recognized by 5 脳 His monoclonal antibody (mouse) antigen. The recombinant protein could be recognized by rabbit polyclonal antibody against Schistosoma japonicum, and when the induced expression time was 4 h, the final concentration of IPTG was 1.0 mmol / L, the expression level was the highest. Conclusion: the prokaryotic expression system of Sj31 gene was successfully constructed, and the recombinant protein peptide had good antigenicity, which laid a necessary foundation for further study of its function.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R383.2

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