科尔沁牛乳腺炎临床分离无乳链球菌菌毛岛屿PI-2a辅助蛋白2基因的克隆及序列分析

Cloning and sequencing of PI-2a fimbriae island ancillary protein 2 gene of bovine mastitis Streptococcus agalactiae from Horqin diary cattles

目的克隆内蒙古东部地区科尔沁牛乳腺炎无乳链球菌菌毛岛屿PI-2a辅助蛋白2(ancillary protein 2,AP2)基因片段,并进行序列分析。方法根据Gen Bank中登录的牛乳腺炎无乳链球菌菌毛岛屿PI-2a AP2基因序列,使用生物信息学软件Primer 5.0设计1对特异性引物,以内蒙古东部地区乳源性无乳链球菌临床菌株总DNA为模板,扩增AP2基因片段,与p MD18-T Vector连接,转化至感受态E.coli JM109中,提取质粒,进行PCR及单、双酶切鉴定;将质粒p MD18-T-AP2转化感受态E.coli JM109后,利用DNAStar分析测序结果,并对其推导的氨基酸序列进行抗原可及性分析,Prot Scale在线程序分析其疏(亲)水性。结果质粒p MD18-T-AP2经PCR及单、双酶切鉴定,证明构建正确;扩增的AP2基因序列长度为699 bp,编码233个氨基酸残基,与Gen Bank中登录的无乳链球菌菌毛岛屿PI-2a AP2基因核苷酸序列相似性达99%,有3个碱基出现变异(402:C→A;563:C→T;467:A→T),氨基酸序列有两处出现变异,分别为178氨基酸(V→A)和216氨基酸(E→V);该序列二级结构α螺旋分布C-末端较多,β折叠较丰富,分布较均匀,无规则卷曲分布在两端,亲水性指数相对较高,抗原性较好。结论成功克隆出内蒙古东部地区科尔沁牛乳腺炎无乳链球菌菌毛岛屿PI-2a AP2基因,为进一步研究AP2基因编码蛋白的抗原性奠定了基础。

Objective To clone and sequence the PI-2a fimbriae island ancillary protein 2（AP2）gene of bovine mastitis Streptococcus agalactiae from Horqin diary cattles in east part of Inner Mongolia Autonomous Region, China. Methods According to the published PI-2a fimbriae island AP2 gene sequences of bovine S. agalactiae in Gen Bank, a pairs of specific primers were designed by bioinformatics software of Primer 5. 0, based on which AP2 gene sequence was amplified using the total DNA of the S. agalactiae clinical strains from the milk of Horqin diary cattles in east part of Inner Mongolia Autonomous Region, and inserted into p MD18-T vector. The constructed recombinant plasmid was transformed to competent E. coli JM109, from which the plasmid was extracted and identified by PCR and restriction analysis.Recombinant plasmid p MD18-T-AP2 was transformed to competent E. coli JM109, analyzed for sequencing result by DNAStar software. The deduced amino acid sequence was analyzed for antigen accessibility, then for hydrophily /hydrophobicity by Prot Scale online program. Results PCR and restriction analysis proved that recombinant plasmid p MD18-T-AP2 was constructed correctly. The amplified AP2 gene, at a length of 699 bp, encoded 233 amino acid residues, of which the homology of nucleotide sequence was 99% to that of PI-2a AP2 gene of S. agalactiae in Gen Bank.Variations were observed in three bases, i. e. C →A at site 402, C →T at site 563 and A →T at site 467. However, two variations were observed in amino acid sequence, i. e. V→A at site 178 and E→V at site 216. In the secondary structure,more α-helixes appeared at C-terminus, while β-sheets were abundant and distributed evenly, and the irregular coli at both the terminuses. The polypeptide encoded by AP2 gene showed high hydrophily and antigenicity. Conclusion The AP2 gene of PI-2a of bovine mastitis S. agalactiae of east part of Inner Mongolia Autonomous Region, China was successfully cloned, which laid a foundation of further study on the antigenicity of protein encoded by AP2 gene.