拼接信号序列单碱基变异提高马传染性贫血病毒mRNA拼接效率

One Nucleotide Mutation in the Spl icing Acceptor Enhances RNA Splici ng Efficiency in Equine Infectious Anemia Virus

分别以马传染性贫血(马传贫)驴强毒(D-AEIAV)RNA和马传贫驴白细胞弱毒疫苗(DLAEIAV)RNA为模板,利用RT-PCR的方法,克隆到马传贫强、弱毒株基因组外显子-2及其下游的核苷酸序列。然后将报告基因CAT插入到EIAV内含子2env阅读框架中,构成CAT拼接报告系统。同时在强毒株重组表达质粒的基础上,将其外显子-3上游拼接受体位点的核苷酸序列CAG突变为弱毒株相应位置的核苷酸序列TAG,得到强毒单核苷酸突变株重组表达质粒。用构建的3个重组表达质粒DNA转染驴血白细胞,ELISA检测转染细胞CAT浓度。结果表明:EIAV强毒株重组表达质粒中CAT蛋白表达量最高,EIAV强毒株重组表达质粒次之,EIAV强毒突变株重组表达质粒最低。由于CAT基因被插入于各重组质粒中的EIAV内含子-2里,EIAV外显子-2、3之间的拼接可导致该基因的删除,因而其拼接效率低于EIAVmRNA外显子-2、3之间的拼接效率。实验数据表明,EIAVSA2拼接信号序列单碱基变异提高了SD2-SA2拼接效率;D-AEIAVSA2-SD2拼接效率比DLAEIAV相应位点拼接效率高。

In this study,viral RNAs were extracted from donkey-adapted equine infectious anemia virus(D-A EIAV)and donkey leukocyte attenuat ed equine infectious anemia virus( DLA EIAV).After the exon2 and its downstream nucleotide sequences of D-A EIAV and DLA EIAV were amplifi ed respectively by RT-PCR,they wer e cloned into pCDNA3 vector.D-A mu tant construct was made by mutatio n of one base in the splicing acce ptor 2.The reporter gene, CAT gene, was inserted into the intron2 of E IAV of all these reporter construc ts to make a reporting system.The CAT gene was supposed to be delete d by the splicing event between SD 2 and SA2.Therefore the amount of CAT production was reversely correla ted with the splicing efficiency.D onkey leukocytes were transfected w ith the three reporter constructs respectively.Results showed that t he single nucleotide mutation in S A2 splicing signal sequence change d the efficiency of SA2-SD2 splici ng;SA2-SD2 splicing efficiency in D-A EIAV was higher than that of DL A EIAV.