C群脑膜炎奈瑟菌外膜蛋白NhhA重组载体pASK-IBA37plus-nhhA的构建

Construction of Recombinant Vector pASK-IBA37plus-nhhA Encoding Outer Membrane Protein NhhA of Serogroup C Neisseria meningitidis

构建C群脑膜炎奈瑟菌外膜蛋白Nhh A重组载体p ASK-IBA37plus-nhh A。以脑膜炎奈瑟菌C群菌株为实验材料,根据Gene Bank上的已知序列设计引物,通过聚合酶链反应(polymerase chain reaction,PCR)扩增C群脑膜炎奈瑟菌外膜蛋白Nhh A基因(nhh A)。将nhh A与p ASK-IBA37 plus质粒用EcoRI,Xho I进行双酶切并尝试不同条件进行连接;连接产物实现在大肠杆菌TOP10感受态细胞中的转化。使用验证质粒图谱初筛和PCR与EcoRI,Xho I双酶切双重验证法复筛的方式筛选p ASKIBA37plus-nhh A阳性转化子。最后将构建好的p ASK-IBA37plus-nhh A进行基因测序。成功构建C群脑膜炎奈瑟菌外膜蛋白Nhh A重组载体p ASK-IBA37plus-nhh A,同时发现nhh A与p ASK-IBA37plus摩尔数之比为5.1、连接体系20μL、DNA终浓度为1.7 ng/μL时,获得的转化子阳性率能达到18.8%,且转化子菌落总数数量适中,方便后续筛检。同时,测序得到1 797 bp的nhh A序列信息,在Gen Bank上进行序列相似性比对,发现该基因与脑膜炎奈瑟菌NGH38的外膜蛋白GNA992(Neisseria meningitidis strain NGH38 outer membrane protein GNA992)基因的相似性为98%。

To construct recombinant plasmid pASK-IBA37plus-nhhA encoding outer membrane protein NhhA of serogroup C Neisseriameningitidis.The gene of outer membrane protein NhhA of serogroup C Neisseria meningitidis(nhhA)is obtained by PCR technique using chromosomal DNA of serogroup C Neisseriameningitidis as a template.And the primers are designed according to the reported sequence published in GeneBank.The PCR product of nhhA and pASK-IBA37 plus vector are ligated after double digestion with EcoR I and Xho I in different conditions,and then the ligation product is transformed into competent cell of E.coli TOP10.Plasmid profile and plasmid DNA restriction endonuclease analysis(digestion with EcoR I and Xho I)are carried out to screen the positive clones.Then,the recombinant vector pASK-IBA37plus-nhhA is carried out gene sequencing analysis.TThe recombinant vector pASK-IBA37plus-nhhA is constructed successfully.And when the molar ratio of nhhA and pASK-IBA37plus is 5∶1 in the 20μL of Ligation system,and the final concentration of the recombinant vector is 1.7 ng/μL,the proportion of positive clones is about 18.8%,and the colony count is moderate,which is convenient to the subsequent screening.At the same time,compared with the homologous sequence published in GenBank,the homologous rate of the 1 797 bp nhhA DNA fragment obtained by gene sequencing and the gene encoding outer membrane protein GNA992 of Neisseria meningitidis strain NGH38 is 98%.