大鼠血清淀粉样物质P(SAP)基因的克隆及重组腺病毒制备

Cloning of rat serum amyloid P component gene and preparation of its recombinant adenovirus vector

目的:制备能够表达大鼠血清淀粉样物质P(Serum amyloid P component,SAP)的重组腺病毒。方法:采用RT-PCR扩增的方法获取大鼠SAP,并将该基因采用定向克隆的方法克隆进入穿梭质粒pAdTrace-TO4中,进一步采用同源重组的方法将该质粒和腺病毒骨架质粒Adeasy-1在大肠杆菌BJ5183中同源重组,然后用脂质体介导的方法导入包装细胞HEK293中,产生表达目的基因的重组腺病毒,扩增病毒后用倍比稀释法测定其滴度。结果:通过PCR技术从重组穿梭质粒和重组腺病毒质粒中扩增出700bp左右插入片段,测序结果和基因库中的基因序列一致。扩增后的重组腺病毒滴度为2.8×108pfu/ml。结论:成功构建了携带SAP基因的重组腺病毒载体Adeasy-1-SAP,为探讨SAP对肺纤维化的作用及其机制打下基础。

Objective：To construct the recombinant adenovirus vector carrying rat serum amyloid P component（SAP）as preparation for later use.Methods：Rat SAP was obtained by using RT-PCR amplification and then cloned into the shutter plasmid pAdTrace-TO4.Subsequently,this newly constructed plasmid pAdTrace-TO4-SAP was followed by homologous recombination with bone plasmid pAdEasy-1 in BJ5183.Recombinant adenovirus was obtained after being packaged in human embryonic kidney cells HEK293 by lipofectAmineTM2000 mediation.After amplification the recombined adenovirus,we determined its titer by end-point dilution assay.Results：In recombinant shuttle plasmid and recombinant adenovirus,a fragment of 700 bp detected by PCR was identical with that included in Genbank.The titer of recombinant adenovirus reached 2.8×10^8 pfu/ml.Conclusion：The recombinant adenovirus containing SAP gene was constructed successfully.This will provides a good basis for further study about the influence of SAP on lung fibrosis.