猪蛔虫抗菌肽Cecropin p基因的克隆及其原核表达载体的构建

The Cloning and the Construction of Expression Vector of Cecropin p from Ascaris Suum

从猪蛔虫提取总RNA,反转录成cDNA后分别设计引物对Cecropin 2、Cecropin 3和Cecropin 4基因进行PCR扩增,分别得到带有双酶切位点的目的基因片段.将目的基因片段与PMD18 T载体连接,转化Top10克隆菌,测序正确后克隆入pET28a表达载体,进行双酶切和测序鉴定.研究结果表明,克隆得到的Cecropin p2、Cecropin p3和Cecropin p4基因均由225个碱基组成,一个编码由74个氨基酸残基组成的开放阅读框,其蛋白相对分子质量约为8 kD,等电点分别为9.86、10.16和9.16.同源性分析结果显示:克隆所得猪蛔虫Cecropin p2、Cecropin p3和Cecropin p4与数据库NCBI中的猪蛔虫Cecropin p2、Cecropin p3和Cecropin p4基因同源性均为100%,与其它Cecropin p基因同源性也较高(>42%).分子进化树显示,Cecropinp4的分化出现最早,其次为Cecropin p2,Cecropin p1和Cecropin p3分化得最晚.成功克隆了猪蛔虫Cecropin 2、Cecropin 3和Cecropin 4基因,构建了它们的原核表达载体并进行了生物信息学分析,为其重组表达和抑菌活性鉴定等研究奠定了理论基础.

Extracted the RNA from Ascaris suum and reverse-transcribed into cDNA.Ran PCR amplification and linked to PMD18 T vector,then transformed into E.coli Top10 and went on sequencing.cloned the gene into pET28a expression vector and made Restriction enzymes digestion analysis.The sequencing showed the cloned Cecropin 2,Cecropin 3,Cecropin 4 genes from Ascaris suum all contain 225 bp encoding an open reading frame of 74 amino acids with the deduced molecular weight around 8 kD and the isoelectric point 9.86、10.16、9.16 respectively.homology analysis indicated All 3 genes are of high homology with the sequences of corresponding Cecropin p genes registered on NCBI,and the differentiation of Cecropin p4 preceded that of the others showed by phylogenic tree.The genes of Cecropin2,Cecropin3 and Cecropin4 were cloned successfully,expression vectors were constructed and the sequence analysis including Homological analysis,phylogenic tree were done,all of these has paved the way for the protein expression and the test of antimicrobial activities.