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利用RT-PCR扩增马铃薯卷叶病毒(PLRV)外壳蛋白(CP)基因(PLRV-CP),回收大小约630bp的特异性扩增片段并进行T-A克隆,测序表明该基因长度为627bp,与已报道的36个PLRV-CP基因的核苷酸序列的同源性大于96%。以pBAD/Thio-TOPO为起始载体,构建了PLRV-CP基因的原核表达载体pBAD-LRCP。以pBAD-LRCP为模板,用PCR法删除了该基因富含精氨酸稀有密码子的第52～177核苷酸,获得了PLRV缺失突变CP基因的原核表达载体pBAD-LRCP-126。用阿拉伯糖诱导工程菌TOP10(pBAD-LRCP-126),获得了34kD的诱导表达的融合蛋白(重组CP)。用镍离子亲和层析法从包涵体中纯化出了高纯度的重组CP,用纯化的重组CP作抗原免疫家兔获得了PLRV特异性的抗血清,间接ELISA检测显示效价为1︰12800。本研究结果为利用重组CP作抗原大量制备PLRV抗血清奠定了基础。
The PLRV-CP gene was amplified by RT-PCR and the specific amplified fragment of 630bp was recovered and TA cloned. Sequencing showed that the gene was 627bp in length, The nucleotide sequence homology of the reported 36 PLRV-CP genes was greater than 96%. Using pBAD / Thio-TOPO as the starting vector, a prokaryotic expression vector pBAD-LRCP of PLRV-CP gene was constructed. Using pBAD-LRCP as a template, we deleted the 52-77th nucleotide of arginine rare codons in this gene by PCR and obtained the prokaryotic expression vector pBAD-LRCP-126 of CP gene with deletion mutation of PLRV. Induction of the engineered bacteria TOP10 (pBAD-LRCP-126) with arabinose resulted in the induction of a 34 kD fusion protein (recombinant CP). High purity recombinant CP was purified from inclusion bodies by nickel ion affinity chromatography. PLRV-specific antiserum was obtained by immunizing rabbits with the purified recombinant CP as an antigen. The indirect ELISA assay showed that the titer was 1︰12800 . The results of this study laid the foundation for the preparation of PLRV antisera using recombinant CP as antigen.