摘要
目的构建汉坦病毒HTN型和SEO型S基因毕赤酵母表达系统。方法应用RT-PCR法扩增汉坦病毒Z10株和L99株编码核蛋白的S基因,将扩增产物分别与pGEM-T-Easy载体连接,经序列分析后双酶切,分别定向克隆入载体pPICZαΑ和pPICZαC,继而将重组质粒以SacⅠ线性化并电转化入毕赤酵母X33感受态细胞,用Zeocin筛选高拷贝转化子进行鉴定。结果PCR扩增产物约为1290bp,与T载体相连测序结果与Genbank相应序列比较,核苷酸序列同源性分别为99.84%和99.92%,其编码蛋白质二级结构均无改变。定向克隆获得pPICZα-ΑZ10S和pPICZ-αL99S,分别电转化得到巴氏毕赤酵母重组菌株PpX33-Z10S和PpX33-L99S,提取其基因组DNA经PCR鉴定与预期相符。结论成功构建汉坦病毒HIN型和SEO型S基因毕赤酵母表达系统,为进一步研究奠定基础。
To construct the S-segment of Hantavirus and Seoul virus in Pichia pastoris expression system, the S-genes of the Z10 and L99 strains of Hantaan virus encoding for nucleoprotein were amplified by RT-PCR, and then the RT-PCR products were cloned into the pGEM-T-Easy vector. After identifying by sequencing analysis, the foreign gene in the recombinant plasmid was cut by restriction enzymes and inserted into plasmids pPICZo.A and pPICZaC respectively, Meanwhile, after linerized by Sac I enzyme, the recombinant plasmid was transformed to the competent Pichia pastoris X33 cells, and the multi-copy transforrnants were screened with Zeocin, It was found that the size of the RT-PCR-product was about 1290 bp. The similarities in nucleotide sequences of this gene in comparison with the sequence of the S-segment of gene inserted into the pGEM-T-Easy vector with the corresponding sequence reported in Genbank were 99,84 % and 99.92 % respectively. In addition, the secondary structures of protein encoded showed no any change. When the recombinant plasmid pPICZaA-Z10S and pPICZaC-L99S were transformed into Pichia pastoris, the recombinant strains of PpX33-Z10S and PpX33-L99S were obtained, and their genomic DNA was proved to be corrected after identification with PCR. It is evident that the expression system in Pichia pastoris was successfully constreted as reported in the present study, thus proriding a useful model of further studies.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2006年第1期11-14,共4页
Chinese Journal of Zoonoses
基金
天津市自然科学基金资助项目(No.033605311)
天津市科技发展计划项目(No.043113511)
