本文選題：肺炎鏈球菌 + 肺炎鏈球菌脫毒溶血素��； 參考：《中國新藥雜志》2017年11期

【摘要】：目的:對2種肺炎鏈球菌蛋白肺炎鏈球菌脫毒溶血素(detoxified pneumolysin,PLYd)和肺炎鏈球菌表面黏附素(pneumococcal surface adhesin A,Psa A)進(jìn)行全基因合成、重組表達(dá)、制備和鑒定。方法:根據(jù)Gen Bank中PLYd,Psa A的基因序列和氨基酸序列,通過密碼子優(yōu)化和全基因合成的方式獲得2種蛋白的基因,在PLY基因中引入多位點(diǎn)突變保證表達(dá)的蛋白無毒性,構(gòu)建無標(biāo)簽、高效重組表達(dá)菌株,建立2種重組蛋白的純化制備方法,并進(jìn)行抗原鑒定及免疫原性檢測。結(jié)果:人工合成的PLYd,Psa A基因序列經(jīng)鑒定與預(yù)期一致。2種蛋白在大腸桿菌中均獲得了高效表達(dá),表達(dá)量在40%以上,經(jīng)2步純化其純度均達(dá)到95%。2種蛋白均能與陽性血清產(chǎn)生特異性抗原抗體反應(yīng),免疫小鼠后均可誘導(dǎo)產(chǎn)生較高水平的蛋白特異性抗體。結(jié)論:成功制備2種無標(biāo)簽肺炎鏈球菌蛋白PLYd,Psa A,且具備與天然抗原相似的抗原性和免疫原性,為開展應(yīng)用研究奠定基礎(chǔ)。
[Abstract]:Objective: to synthesize, express, prepare and identify two strains of Streptococcus pneumoniae protein detoxified pneumolysin plyd and pneumococcal surface adhesin PSAA. Methods: according to the gene sequence and amino acid sequence of PLYdSSA A in GenBank, two kinds of protein genes were obtained by codon optimization and gene synthesis. Two methods of purification and preparation of recombinant protein were established, and antigen identification and immunogenicity detection were carried out. Results: the synthetic PLYdfen Psa A gene sequence was confirmed to be in accordance with the expectation. 2 proteins were highly expressed in Escherichia coli, and the expression level was over 40%. After two steps of purification, the purity of 95. 2 proteins could react with the positive serum to produce specific antigens and antibodies, which could be induced to produce a higher level of protein specific antibodies after immunizing mice. Conclusion: two kinds of unlabeled streptococcus pneumoniae protein PLYdN Psa A were successfully prepared with antigenicity and immunogenicity similar to those of natural antigens, which laid the foundation for application research.
【作者單位】： 中國食品藥品檢定研究院;
【基金】：國家高技術(shù)研究發(fā)展計劃(863計劃)(2012AA02A402)
【分類號】：R945

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