甘忠桥;孟祥玉;李博;陈晓瑞;张悦;袁剑文;梁丹虹;吴永革;谷铁军;

• 1:吉林大学艾滋病疫苗国家工程实验室

摘要(Abstract)：

目的优化肺炎疫苗重组蛋白基因Psa A-Psp A23的同源区序列,阻止该基因的组氨酸标签(His-tag)融合表达,对突变后的目的基因进行表达及鉴定。方法以肺炎疫苗重组蛋白基因Psa A-Psp A23为模板,设计2对具有重叠区的特异性引物,并分别对该重组基因进行扩增,得到Psa A-Psp A2和Psp A3的CDR区(亚类决定区)基因片段。利用重叠延伸PCR(Overlap PCR)技术,将Psa A-Psp A2基因片段和Psp A3的CDR区基因片段连接,得到同源区优化后的基因片段。利用PCR技术扩增同源区优化后的基因片段,使该基因3′末端引入终止密码子,阻止Psa APsp A23基因表达载体上His-tag的融合表达。将得到的片段同源区优化且无His-tag融合表达的目的基因连接至p ET20b载体后,转入大肠埃希菌(E.coli)中进行表达,表达产物进行Western blot分析。结果重组表达质粒p ET20b-Psa A-Psp A23经双酶切和测序鉴定,证明构建正确;转化E.coli中,37℃,IPTG终浓度1 mmol/L诱导5 h,获得可溶性表达的重组蛋白,且无His-tag。结论 Psa A-Psp A23蛋白基因同源区优化及His-tag成功去除,并在原核表达系统E.coli中可溶性表达,可为Psa A-Psp A23蛋白疫苗的进一步研究奠定基础。

关键词(KeyWords)： 组氨酸标签;肺炎链球菌表面蛋白A;同源区序列;重叠延伸PCR

Abstract:

Keywords:

基金项目(Foundation):

作者(Authors): 甘忠桥;孟祥玉;李博;陈晓瑞;张悦;袁剑文;梁丹虹;吴永革;谷铁军;

DOI: 10.13200/j.cnki.cjb.002276

参考文献(References)：

• [1]DENG X,CHURCH D,VANDERKOOI O G,et al.Streptococcus pneumoniae infection:a Canadian perspective[J].Expert Rev Anti infect Ther,2013,11(8):781-791.
• [2]WALKER C L F,RUDAN I,LIU L,et al.Global burden of childhood pneumonia and diarrhoea[J].Lancet,2013,381(9875):1405-1416.
• [3]JACKSON L A,GURTMAN A,VAN CLEEFF M,et al.Immunogenicity and safety of a 13-valent pneumococcal conjugate vaccine compared to a 23-valent pneumococcal polysaccharide vaccine in pneumococcal vaccine-naive adults[J].Vaccine,2013,31(35):3577-3584.
• [4]SANTAMARIA R,GOULART C,PERCIANI C T,et al.Humoral immune response of a pneumococcal conjugate vaccine:capsular polysaccharide serotype 14-Lysine modified Psp A[J].Vaccine,2011,29(47):8689-8695.
• [5]YOTHER J,WHITE J M.Novel surface attachment mechanism of the Streptococcus pneumoniae protein Psp A[J].J Bacteriol,1994,176(10):2976-2985.
• [6]YU J,LI B,CHEN X,et al.Comparison of immunogenicity and protection of two pneumococcal protein vaccines based on Psa A and Psp A[J].Infect Immun,2018,86(6).pii:e00916-17.doi:10.1128/IAI.00916-17.
• [7]LU J,SUN T,WANG D,et al.Protective immune responses elicited by fusion protein containing Psa A and Psp A fragments[J].Immunol Invest,2015,44(5):482-496.
• [8]VASHISHTA M,KHAN N,MEHTO S,et al.Pneumococal surface protein A(Psp A)regulates programmed death ligand 1expression on dendritic cells in a Toll-like receptor 2 and calcium dependent manner[J].PLo S one,2015,10(7):e013-3601.doi:10.1371/journal.pone.0133601.
• [9]KNUPP C,LUTHER P K,SQUIRE J M.Titin organisation and the 3D architecture of the vertebrate-striated muscle I-band[J].J Mol Biol,2002,322(4):731-739.
• [10]张恒铭,公雪杰.疫苗自身免疫毒性评价法规现状[C].第五次全国免疫诊断暨疫苗学术研讨会论文集,宁夏银川,2011:483-484.
• [11]XIE H,YANG C,CHEN L E.Current strategies for polypeptide fusion tags removal[J].Prog in Biochem Biophysics,2009,36(10):1364-1369.(in Chinese)谢浩,杨程,陈丽娥.多肽融合标签的移除策略[J].生物化学与生物物理进展,2009,36(10):1364-1369.
• [12]TAN L,CHEN H,YU S,et al.A SOE-PCR method of introducing multiple mutations into Mycoplasma gallisepticum neuraminidase[J].J Microbiol Methods,2013,94(2):117-120.
• [13]WANG Z J,LI H M,GONG A J,et al.Research advances of separation and purification for protein[J].Chem&Bioengi,2009,26(8):8-11.(in Chinese)王子佳,李红梅,弓爱君,等.蛋白质分离纯化方法研究进展[J].化学与生物工程,2009,26(8):8-11.