本文選題：淋球菌 + 免疫血清　； 參考：《大理學(xué)院》2010年碩士論文

【摘要】：研究目的 制備淋球菌免疫血清,用于Western Blot技術(shù)中以檢測(cè)NspA重組蛋白的表達(dá)情況；構(gòu)建淋球菌奈瑟氏表面蛋白A(Neisseria surface protein A, NspA)基因原核表達(dá)載體,并誘導(dǎo)NspA融合蛋白在宿主菌BL21 (DE3)中表達(dá),為淋球菌NspA蛋白的進(jìn)一步研究提供基礎(chǔ)。 研究方法 淋球菌免疫血清的制備：制備滅活淋球菌菌體、淋球菌裂解物兩種免疫原,免疫家兔后收獲免疫血清。用于Western Blot技術(shù)中以檢測(cè)NspA重組蛋白的表達(dá)情況。 構(gòu)建pGEX4T-1-nspA原核表達(dá)載體并誘導(dǎo)表達(dá)：根據(jù)基因庫報(bào)道的淋球菌nspA基因序列,設(shè)計(jì)合成一對(duì)引物；以淋球菌基因組DNA為模板,PCR擴(kuò)增淋球菌nspA基因；將PCR擴(kuò)增產(chǎn)物與帶有谷胱甘肽S-轉(zhuǎn)移酶(glutathione S-tramsferases GSTs)基因的高效原核表達(dá)質(zhì)粒pGEX4T-1定向重組,構(gòu)建原核表達(dá)重組體pGEX4T-1-nspA;重組子經(jīng)酶切、PCR鑒定和核酸序列分析正確后,異丙基-β-D-硫代半乳糖苷(IPTG)誘導(dǎo)重組蛋白表達(dá),用SDS-PAGE及Western blot技術(shù)檢測(cè)重組蛋白表達(dá)情況。 結(jié)果 成功制備淋球菌免疫血清：制備的兩組免疫血清效價(jià)均達(dá)到1：12800以上,并將其用于Western Blot檢測(cè)重組蛋白的表達(dá),在約44KDa位置出現(xiàn)特異性結(jié)合條帶； 成功構(gòu)建pGEX4T-1-nspA原核表達(dá)載體并誘導(dǎo)表達(dá)以淋球菌基因組DNA為模板,通過PCR擴(kuò)增,獲得淋球菌nspA基因；經(jīng)雙酶切、PCR和核酸序列分析表明：成功構(gòu)建淋球菌nspA基因原核表達(dá)重組體pGEX4T-1-nspA; SDS-PAGE及Western blot分析顯示：重組nspA基因在原核系統(tǒng)中得到了表達(dá),融合蛋白大小約44kDa。 結(jié)論 成功制備兔抗淋球菌免疫血清,并能特異性識(shí)別NspA重組蛋白并與之結(jié)合,為本研究及后續(xù)的淋球菌其他研究提供準(zhǔn)備； 成功構(gòu)建pGEX4T-1-nspA原核表達(dá)載體,并在大腸桿菌中表達(dá),為研究NspA蛋白的生物活性、淋球菌動(dòng)物免疫實(shí)驗(yàn)、淋球菌檢測(cè)及其作為粘膜免疫疫苗的研究等應(yīng)用價(jià)值奠定基礎(chǔ)。
[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.
【學(xué)位授予單位】：大理學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R346

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