【摘要】： 目的:克隆細粒棘球絳蟲Eg95、EgA31抗原基因,構建pET30a-EgA31及pET30a- EgA31-Eg95原核表達載體,并在大腸埃希菌宿主系統(tǒng)中表達EgA31及EgA31-Eg95重組蛋白,對表達產物進行免疫印跡分析。方法:從細粒棘球絳蟲原頭蚴及成蟲組織中提取總RNA,反轉錄生成cDNA,分別以原頭蚴cDNA及成蟲cDNA為模板RT-PCR克隆獲得Eg95和EgA31抗原基因,將其克隆至pUCm-T載體,測序確定其正確性。利用定向克隆技術將EgA31抗原基因片段克隆至原核表達質粒pET30a上,轉化E.coli DH5α,根據選擇標記的卡那霉素抗性基因篩選到陽性克隆,通過PCR分析和酶切鑒定篩選出陽性克隆,轉化BL2(1DE3),IPTG初步誘導表達pET30a-EgA31重組蛋白,SDS-PAGE電泳檢測,凝膠圖像分析系統(tǒng)確定目的蛋白表達水平。以測序正確的pET30a-EgA31重組質粒為載體,運用酶切連接技術構建pET30a-EgA31 -Eg95重組質粒,表達pET30a-EgA31-Eg95重組蛋白,SDS-PAGE電泳檢測,并經凝膠圖像分析確定目的蛋白的表達水平。結果:測序表明選取的pET30a-EgA31及pET30a-EgA31-Eg95陽性克隆均為正確連接目的基因的重組質粒。經IPTG誘導后重組蛋白均得到成功表達,分別在相對分子量約為31 kDa及46 kDa處有表達條帶,表達量約占菌體總蛋白質的26%及20%。結論:成功克隆并構建了pET30a-EgA31及pET30a-EgA31-Eg95原核表達質粒,初步誘導表達出EgA31、EgA31-Eg95及Eg95重組蛋白,為進一步研究其免疫特性奠定了基礎。
[Abstract]:Aim: to clone the Eg95,EgA31 antigen gene of Echinococcus granulosus, construct the prokaryotic expression vector of pET30a-EgA31 and pET30a- EgA31-Eg95, and express EgA31 and EgA31-Eg95 recombinant proteins in the host system of Escherichia coli. Methods: Eg95 and EgA31 antigenic genes were cloned from Echinococcus granulosus (Echinococcus granulosus) protocaria and adult Echinococcus granulosus (Echinococcus granulosus) tissues by reverse transcription of total RNA, to generate cDNA,. Eg95 and EgA31 antigenic genes were cloned into pUCm-T vector using cDNA and cDNA as templates, respectively. Sequencing confirmed its correctness. The EgA31 antigen gene fragment was cloned into prokaryotic expression plasmid pET30a by directional cloning technique, and transformed into E.coli DH5 偽. The positive clones were screened by selective marker kanamycin resistance gene. The positive clones were screened by PCR analysis and restriction endonuclease analysis. Transforming BL2 (1DE3), IPTG induced expression of pET30a-EgA31 recombinant protein), SDS-PAGE electrophoresis detection, gel image analysis system to determine the expression level of the target protein. The recombinant plasmid of pET30a-EgA31-Eg95 was constructed by restriction endonuclease ligation with the pET30a-EgA31 recombinant plasmid which was sequenced correctly. The recombinant pET30a-EgA31-Eg95 protein was expressed and detected by SDS-PAGE electrophoresis. The expression level of the target protein was determined by gel image analysis. Results: sequencing showed that the selected pET30a-EgA31 and pET30a-EgA31-Eg95 positive clones were recombinant plasmids with correct ligation of the target gene. After induction by IPTG, the recombinant protein was successfully expressed, and the relative molecular weight was about 31 kDa and 46 kDa, respectively. The expression amount of the recombinant protein was about 26% and 20% of the total cell protein, respectively. Conclusion: the prokaryotic expression plasmids of pET30a-EgA31 and pET30a-EgA31-Eg95 were cloned and constructed successfully, and the recombinant proteins of EgA31,EgA31-Eg95 and Eg95 were induced and expressed, which laid a foundation for further study of their immunological characteristics.
【學位授予單位】：新疆醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R392

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