淋球菌nspA基因原核重组质粒的构建及表达

发布时间：2018-05-29 14:18

本文选题：淋球菌 + 免疫血清　；参考：《大理学院》2010年硕士论文

【摘要】：研究目的制备淋球菌免疫血清,用于Western Blot技术中以检测NspA重组蛋白的表达情况；构建淋球菌奈瑟氏表面蛋白A(Neisseria surface protein A, NspA)基因原核表达载体,并诱导NspA融合蛋白在宿主菌BL21 (DE3)中表达,为淋球菌NspA蛋白的进一步研究提供基础。研究方法淋球菌免疫血清的制备：制备灭活淋球菌菌体、淋球菌裂解物两种免疫原,免疫家兔后收获免疫血清。用于Western Blot技术中以检测NspA重组蛋白的表达情况。构建pGEX4T-1-nspA原核表达载体并诱导表达：根据基因库报道的淋球菌nspA基因序列,设计合成一对引物；以淋球菌基因组DNA为模板,PCR扩增淋球菌nspA基因；将PCR扩增产物与带有谷胱甘肽S-转移酶(glutathione S-tramsferases GSTs)基因的高效原核表达质粒pGEX4T-1定向重组,构建原核表达重组体pGEX4T-1-nspA;重组子经酶切、PCR鉴定和核酸序列分析正确后,异丙基-β-D-硫代半乳糖苷(IPTG)诱导重组蛋白表达,用SDS-PAGE及Western blot技术检测重组蛋白表达情况。结果成功制备淋球菌免疫血清：制备的两组免疫血清效价均达到1：12800以上,并将其用于Western Blot检测重组蛋白的表达,在约44KDa位置出现特异性结合条带；成功构建pGEX4T-1-nspA原核表达载体并诱导表达以淋球菌基因组DNA为模板,通过PCR扩增,获得淋球菌nspA基因；经双酶切、PCR和核酸序列分析表明：成功构建淋球菌nspA基因原核表达重组体pGEX4T-1-nspA; SDS-PAGE及Western blot分析显示：重组nspA基因在原核系统中得到了表达,融合蛋白大小约44kDa。结论成功制备兔抗淋球菌免疫血清,并能特异性识别NspA重组蛋白并与之结合,为本研究及后续的淋球菌其他研究提供准备；成功构建pGEX4T-1-nspA原核表达载体,并在大肠杆菌中表达,为研究NspA蛋白的生物活性、淋球菌动物免疫实验、淋球菌检测及其作为粘膜免疫疫苗的研究等应用价值奠定基础。
[Abstract]:Research purpose A prokaryotic expression vector of Neisseria gonorrhoeae (Neisseria gonorrhoeae) surface protein A(Neisseria surface protein A, NspA) gene was constructed, and the NspA fusion protein was induced to express in the host strain BL21 DE3. It provides a basis for further study of NspA protein of Neisseria gonorrhoeae. Research method Preparation of Neisseria gonorrhoeae: preparation of inactivated Neisseria gonorrhoeae, two kinds of immunogen of gonococcal lysates, after immunizing rabbits, the immune serum was harvested. To detect the expression of NspA recombinant protein by Western Blot. Construction and induction of pGEX4T-1-nspA prokaryotic expression vector: a pair of primers were designed and synthesized according to the nspA gene sequence of Neisseria gonorrhoeae reported by gene bank, and the nspA gene of Neisseria gonorrhoeae was amplified by PCR using genomic DNA of Neisseria gonorrhoeae as template. The PCR amplification product and the high efficient prokaryotic expression plasmid pGEX4T-1 containing glutathione S-tramsferases (GSTs) gene were recombined to construct the prokaryotic expression recombinant pGEX4T-1-nspA, and the recombinant plasmid was identified by restriction endonuclease digestion and the nucleic acid sequence analysis was correct. Isopropyl- 尾 -D- thiogalactoside (IPTG) induced the expression of recombinant protein, and the expression of recombinant protein was detected by SDS-PAGE and Western blot techniques. Result Preparation of Neisseria gonorrhoeae immune serum: the titers of the two groups were above 1: 12800, and they were used to detect the expression of recombinant protein by Western Blot, and there were specific binding bands in about 44KDa position. The prokaryotic expression vector of pGEX4T-1-nspA was successfully constructed and the genomic DNA of Neisseria gonorrhoeae was induced to be expressed as template. The nspA gene of Neisseria gonorrhoeae was amplified by PCR. NspA gene prokaryotic expression recombinant pGEX4T-1-nspA of Neisseria gonorrhoeae was successfully constructed, and SDS-PAGE and Western blot analysis showed that the recombinant nspA gene was expressed in the prokaryotic system, and the fusion protein was about 44kDa. Conclusion Rabbit anti-gonococcal immune serum was successfully prepared, and NspA recombinant protein was specifically recognized and combined with it, which provided preparation for this study and other studies on gonococcus. The prokaryotic expression vector of pGEX4T-1-nspA was successfully constructed and expressed in Escherichia coli, which laid a foundation for the study of the biological activity of NspA protein, the animal immunoassay of Neisseria gonorrhoeae, the detection of Neisseria gonorrhoeae and its application as mucosal immunity vaccine.
【学位授予单位】：大理学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346

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