甘薯脉花叶病毒外壳蛋白基因在大肠杆菌中的表达及其抗血清的制备

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

根据已报道的甘薯脉花叶病毒（Sweet potato vein mosaic virus，SPVMV）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SPVMV河南分离物（SPVMV—HN）基因组3’端1．8kb的基因片段，包括部分NIb基因序列和完整的CP基因及3’端非编码区序列（3’UTR）。序列分析表明，SPVMV—HN的CP基因由996个核苷酸组成（GenBank登录号为FJ687211），编码332个氨基酸残基。与已发表的SPVMV其他分离物相比，其推导的氨基酸序列一致性为95．2％～98．5％，与SPVMV广东分离物的氨基酸序列一致性为97．9％。将CP基因克隆到原核表达载体pET-28a（＋）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SPVMV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。利用SPVMV的抗血清，对采自全国14个省（市）的田间甘薯样品以及嫁接的巴西牵牛样品进行了检测，结果表明，SPVMV在我国甘薯上普遍存在。

According to published nucleotide sequence, the primers were designed. The region of 3＇-terminal 1 800 nts encompassing the part of NIb gene, coat protein （CP） gene and 3＇ untranslated region （UTR） of Sweet potato vein mosaic virus （SPVMV） isolated from Henan Province was cloned and sequenced by RT- PCR. The sequencing showed that the CP gene was consisted of 996 nt and encoded 332 amino acid residues （FJ687211）. The deduced amino acid sequence of CP gene was 95.2% -98.5% identical to other isolates published before, and was 97.9% identical to SPVMV-GD （AY459611） isolate. The CP gene was cloned in- to expression vector pET-28a（ ＋ ）for over-expression in prokaryotic cells. The SDS-PAGE showed that about 41 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 could be used for specific detection of SPVMV from field samples of sweet potato with ACP-ELISA. This antiserum was used for detec- ting the samples of Ipomoea batatas and L setosa inoculated with the field specimens from 14 provinces. The results indicated that the SPVMV was common in China.