犬贾第虫病毒转染载体介导的锤头状核酶对KRR1基因体外转录体切割效果的研究

The Cleavage Activity of GCV Transfer Vector-mediated Hammerhead Ribozyme for KRR1 in vitro Transcript

目的检测犬贾第虫病毒介导的锤头状核酶对犬贾第虫滋养体核仁功能性蛋白KRR1基因体外转录体切割效率。方法将两端携带反义KRR1基因的锤头状核酶(ribozyme,R酶)插入犬贾第虫病毒(Giardia canis virus,GCV)转染载体中,构建两个核酶嵌合型犬贾第虫病毒载体:短反义序列,上游及下游均为21个碱基,形成重组质粒KRzS;长反义序列,上游为288个碱基,下游为507个碱基,形成重组质粒KRzL。另设两种阴性对照,即重组质粒TRzL(即R酶两端含虫体反义磷酸丙糖异构酶基因,上游为324个碱基,下游为380个碱基)和重组质粒PKR(即犬贾第虫病毒转染载体中仅有KRR1基因的反义片段而无R酶功能区)。应用荧光实时定量RT-PCR法检测嵌合型锤头状核酶体外对KRR1基因mRNA切割活性。结果KRzS、KRzL转录体对KRR1基因体外转录RNA切割效率分别达到74.0%和81.1%。而PKR对KRR1 mRNA反转录的影响较小,RT-PCR效率仅降低12.0%。TRzL对KRR1 mRNA反转录几乎无影响。结论犬贾第虫病毒介导的锤头状核酶对KRR1基因体外转录体能进行有效切割,为以后的体内锤头状核酶对KRR1基因表达抑制试验提供依据。

Objective To detect the cleavage activity of Giardia canis virus （GCV） transfer vector-mediated hammerhead ribozyme for KRR1 in vitro transcript. Methods Giardia,a most primitive eukaryote,has KRR1 protein responsible for ribosome biosythesis. cDNA encoding hammerhead ribozyme flanked with various lengths of antisense RNA was cloned into a viral vector pGCV634/GFP/GCV2174 derived from the genome of GCV,KRzS flanked with 21 nt KRR1 antisense RNA on each arm,or KRzL flanked with 288 nt and 507 nt KRR1 antisense RNA. At the same time,two control groups were established： PKR without the inserted ribozyme,and TRzL flanked with 324 nt and 380 nt triosephosphate isomerase（Tim） antisense RNA. The cleavage activity of GCV transfer vector-mediated hammerhead ribozyme for KRR1 in vitro transcript was then analyzed by absolute real-time quantitative RT-PCR. Results The in vitro cleavage activities on KRR1 mRNA of the two ribozyme KRzS or KRzL were 74.0% and 81.1% respectively by the absolute real-time quantitative RT-PCR. The two control groups,PKR or TRzL,showed no effect on KRR1 mRNA in vitro. Conclusion The GCV transfer vector-mediated hammerhead ribozyme shows a high cleavage activity for KRR1 in vitro transcript,which demonstrates the feasibility of using a viral vector to express a ribozyme targeted at a specific mRNA in Giardia to reduce the expression of a specific gene.