Wade-Evans A M, Woolhouse T, O'Hara R, Hamblin C
AFRC, Institute for Animal Health, Pirbright Laboratory, Woking, Surrey, UK.
J Virol Methods. 1993 Dec 15;45(2):179-88. doi: 10.1016/0166-0934(93)90102-w.
A full-length cDNA clone of genome segment 7 of African Horse Sickness Virus, serotype 9 (AHSV9) was obtained using the PCR technique. The clone was sequenced and found to be 98.27% homologous to the previously published sequence of the equivalent cDNA clone from AHSV4 at the nucleotide level and to exhibit 99.7% identity at the amino acid level. The cDNA clone was transferred to pGEX-2T (Pharmacia), a bacterial expression vector, such that the reading frame of AHSV9 VP7 was continuous with that of the bacterial glutathione-S-transferase (GST) protein, under the control of the bacterial tac promoter. On induction with IPTG a fusion protein consisting of GST and VP7 was synthesised, which was readily purified on a GST-sepharose column (Pharmacia). The fusion protein reacted equally well in an indirect ELISA using monoclonal antibodies specific for AHSV9 VP7 or polyclonal guinea pig antisera raised against AHSV9 infectious sub-viral particles. This protein was also shown to be a suitable substitute for virus antigen, prepared from infected BHK cell extracts, in a competitive ELISA. Antibodies titres recorded for AHSV9 positive and negative horse sera were similar in the competitive ELISA using either bacterial AHSV VP7 or BHK extracted virus as the source of antigen, in combination with monoclonal or polyclonal antibodies, respectively, as the detectors.
利用聚合酶链反应（PCR）技术获得了非洲马瘟病毒9型（AHSV9）基因组第7节段的全长cDNA克隆。对该克隆进行测序后发现，其在核苷酸水平上与先前发表的AHSV4等效cDNA克隆序列的同源性为98.27%，在氨基酸水平上的同一性为99.7%。将该cDNA克隆转移至细菌表达载体pGEX - 2T（Pharmacia公司），使得AHSV9 VP7的阅读框与细菌谷胱甘肽 - S - 转移酶（GST）蛋白的阅读框在细菌tac启动子的控制下保持连续。用异丙基 - β - D - 硫代半乳糖苷（IPTG）诱导后，合成了一种由GST和VP7组成的融合蛋白，该融合蛋白可在GST - 琼脂糖柱（Pharmacia公司）上轻松纯化。在使用针对AHSV9 VP7的单克隆抗体或针对AHSV9感染性亚病毒颗粒产生的豚鼠多克隆抗血清的间接酶联免疫吸附测定（ELISA）中，该融合蛋白的反应效果相同。在竞争ELISA中，该蛋白也被证明是从感染的幼仓鼠肾（BHK）细胞提取物中制备的病毒抗原的合适替代物。在竞争ELISA中，分别以细菌来源的AHSV VP7或BHK提取的病毒作为抗原，与单克隆或多克隆抗体作为检测剂，记录的AHSV9阳性和阴性马血清的抗体滴度相似。
