摘要
为了便于山羊痘病毒(goat pox virus,GPV)活载体疫苗的研究,本试验拟构建能够用于GPV重组的通用转移载体。用DNA合成和PCR方法构建含有启动子p7.5和p11的质粒pTKpp,以绿色荧光蛋白(green fluorescent protein,GFP)为报告基因,构建含有p7.5/p11-GFP表达盒的重组质粒pTKpp-GFP和通用转移质粒pTKpgigp,转染已感染GPV的羔羊睾丸(lamb testis,LT)细胞,观察GFP的表达情况;同时在pTKpgigp中插入外源基因FMDVVP1,构建重组转移质粒pTKpgigp-VP1,与GPV共转染LT细胞,产生并筛选重组GPV,验证外源基因的表达情况。结果显示,重组质粒中的启动子p7.5和p11能够启动GFP的表达;以重组转移质粒pTKpgigp-VP1进行的GPV重组成功获得了rGPV。最终证明通用转移载体pTKpgigp构建成功,能够用于重组GPV的相关研究。
In the study,a universal transfer vector used in goat pox virus recombination was constructed to avail recombinant vaccine of goat pox virus. The plasmid pTKpp contained promoter pT. 5 and pll was constructed by DNA synthesis and PCR amplification. Based on the plasmid pT- Kpp and using green fluorescent protein （GFP） as a report gene,the recombinant plasmids,pTK- pp-GFP and pTKpgigp, containing the expression cassette pT. 5/p11-GFP were constructed and transfected into LT cells infected with GPV previously using liposome. The expression of report gene was observed after the transfection. Meanwhile,inserting the FMDV-VP1 gene into the uni- versal transfer plasmid pTKpgigp, the transfer plasmid pTKpgigp-VP1 was constructed and trans fected into LT cells infected with GPV previously. The rGPVs were formed via homologous re- combination and selected by GFP and gpt marker. The expression of VP1 gene in the rGPV was detected by immunofluorescence. The results showed that the GFP was expressed transiently un- der the control of pT. 5/pll promoters. Furthermore, a rGPV/FMDV-VP1 line was rescued and FMDV VP1 gene was expressed steadily in LT cells. In conclusion, the universal transfer vector pTKpgigp was able to be used in the GPV recombination.
作者
李继东
才学鹏
LI Ji-dong CAI Xue-peng(School of Agriculture, Ningxia University,Yinchuan 750021, Chi na State Key Laboratory of Veterinary Etiological Biology ,Lanzhou Veterinary Research Insti tute , Chinese Academy of Agricultural Sciences, Lanzhou 730046, China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2016年第12期2042-2048,共7页
Chinese Journal of Veterinary Science
基金
宁夏大学自然科学研究基金资助项目(ZR1440)
关键词
山羊痘病毒
转移载体
重组
TK基因
goat pox virus
transfer vector
recombination
tk gene
