甘薯潜隐病毒外壳蛋白基因的克隆、表达及其抗血清的制备

 根据已报道的甘薯潜隐病毒(Sweet potato latent virus,SPLV)外壳蛋白(CP)基因的核苷酸序列合成引物,利用RT-PCR方法克隆了SPLV河南分离物(SPLV-HN)的CP基因及部分3'端非编码区序列,序列分析表明,SPLV-HN CP基因由879个核苷酸组成(GenBank登录号为DQ399862),编码293个氨基酸残基。与GenBank中SPLV-CH(X84011)和SPLV-T(X84012)分离物的核苷酸序列相似性分别为96.8%和93.0%;与日本分离物(E15420)的核苷酸序列相似性为83.6%。将CP基因克隆到原核表达载体pET-30a(+)上,SDS-PAGE分析表明,经IPTG诱导,CP基因在大肠杆菌BL21(DE3)pLysS中得到了高效表达。以表达的蛋白为抗原,免疫家兔,制备了SPLV外壳蛋白的特异性抗血清。ACP-ELISA检测结果表明,制备的抗血清可用于田间甘薯样品的检测。

Cloning,expression of coat protein gene of Sweet potato latent virus in E.coli and preparation of antiserum

According to published nucleotide sequence, coat protein (CP) gene of Sweet potato latent virus (SPLV) isolated from Henan Province was cloned and sequenced. The results of sequencing showed that the CP gene was consisted of 879 nt and encoding 293 amino acid residues. The nucleotide sequence of CP gene was 96.8% and 93.0% identical to SPLV-CH (X84011) and SPLV-T (X84012) isolates respectively, and was 83.6% identical to SPLV-Japan(E15420)isolate. The CP gene was cloned into expression vector pET-30a(+)for overexpression in prokaryotic cells. The result of SDS-PAGE showed that about 33 kDa specific fusion protein was produced after induction by IPTG. The expressed protein was purified from SDS-PAGE and the antiserum against the protein was raised in rabbit. The antiserum was used for specific detection of SPLV from field samples of sweet potato by ACP-ELISA.