华支睾吸虫CsSP16.4基因的克隆、序列分析和功能预测

Cloning, sequence and function analysis of gene encoding the secretory protein CsSP16.4 from Clonorchis sinensis

目的通过筛选cDNA文库识别华支睾吸虫新基因;克隆并构建重组表达载体,为进一步研究其功能及其应用价值奠定基础。方法对华支睾吸虫cDNA文库进行筛选,通过Blastn程序进行序列比对,识别华支睾吸虫新基因,并应用Motifscan,NCBIConservedDomainSearch等程序对其进行结构域分析及进化树分析。结果筛选得到的序列长725bp,应用VecterNTI分析,理论分子量为16.4kDa,理论等电点(pI)为6.44,疏水氨基酸(AILFWV)占36%,带电荷氨基酸(RKHY-CDE)占29%,极性氨基酸(NCQSTY)占28.8%,酸性氨基酸(DE)占8.65%和碱性氨基酸(KR)占9.21%。应用NCBI站点的ORFFinding分析发现其含4个可能的ORF,其中最长的ORF,从第42到第494bp,起始密码为ATG、终止密码为TAG,完整阅读框含450bp,编码150aa。发现该基因有潜在的2个糖激化位点、3个N肉豆蔻酰化位点和1个蛋白激酶C磷酸化位点。结论确定该基因为华支睾吸虫分泌蛋白基因,为作为诊断候选抗原提供了依据。

The new gene from Clonorchis sinensis was identified through the screening with the cDNA library of this parasite and such gene was cloned and constructed for the recombinant expression vector. Meanwhile, the analysis for the structural regions and the phylogenetic tree of the CsSP16.4 protein was conducted with Motifscan, NCB1 conserved domain search protocol and the sequence analysis of the new gene was undertaken by the Blastn protocol. It was demonstrated that the screened sequence of gene encoding the secretory protein CsSP16, 4 was found to be 450 bp in length, and this gene comprised of 2 N-glycosylation sites, 3 n-myrisstoylation sites and one protein kinase C phosphorylation site. The theoretical molecular weight and isoelectrie point （pl） of the CsSP16.4 protein were 16.4 kGa and 6.44 respectively, with 36% of hydrophobic amino aeids（AILFWV）, 29% of charged amino acids （RKHYCDE） ,28.8% of polar amino acids （NCQSTY） ,8.65% of acid amino acids （DE） and 9. 21N of alkaline amino acids（KR）. It is evident that this screened gene should be the responsible gene encoding the secretory protein CsSP16.4 and its gene product can be used as the candidate antigen for serological diagnosis, of C. sinesis infection.