苹果茎痘病毒库尔勒香梨分离物外壳蛋白基因的原核表达

Construction of Prokaryotic Expression Vector of Coat Protein of Apple Stem Pitting Virus in Xinjiang Korla Pear and Its Expression in Escherichia coli

【目的】苹果茎痘病毒为潜隐性病毒,该病毒寄主范围和分布广泛,能侵染多种果树。目前侵染新疆库尔勒香梨,造成了严重的经济损失,研究了解ASPV的功能基因及治病机理,并诱导其表达,制备出高效的血清,将病害的损害度降到最低。【方法】用新疆库尔勒香梨携毒枝条的韧皮部提取苹果茎痘病毒RNA,根据已经公布ASPV(EU095327)核苷酸序列,设计合成1对CP基因的引物,通过RT-PCR扩增出CP基因,将其克隆至表达载体pET-3a中。【结果】经测序表明,目的基因CP已正确地整合至表达质粒中。【结论】重组质粒转化大肠杆菌BL21,在不同时间下诱导均获得了表达,且表达蛋白产物的分子质量大小与预期值一致,并可被特异性抗体所识别。

【Objective】Apple stem pitting virus is a latent virus,the virus has lots of hosts and distributes widely,and in addition,it can infect varieties of fruit trees.At present,Xinjing korla pear has been infected this virus and many trees died of disease,causing serious economic losses.So we must clearly understand the function of genes and the mechanism of treating virus.Inducing its expression and obtaining blood serum effectively,try to minimize the degree of disease damage.【Method】Extracting RNAs from bark of Korla pear infected with Apple stem pitting virus（ASPV）.And according to the nucleotide sequence of ASPV（EU095327） had been reported in Genbank,design the primer of coat protein gene.Using RT-PCR,coat protein gene had been obtained and cloned in expression vector pET-3a.【Result】The result of sequence showed that the coat protein gene has been cloned into vector pET-3a.【Conclusion】The recombinant plasmid pET-3aCP was then transformed into BL21（Escherichia coli）,and induced to express ASPV fusion protein by IPTG at 0.5h,1h,2h,3h,4h respectively,the expression product could react with the specific antibody,and the relative molecular weight of the expression product was identical to the expected value.