JIANG Shi-jin, CUI Zhi-zhong, DING Jia-bo, MENG Shan-shan and YANG Han-chun. Prokaryotic Expression of pp24 Gene of Md11 Strain of Marek’s Disease Virus[J]. Virologica Sinica, 2004, 19(4): 369-372.
Citation:
JIANG Shi-jin, CUI Zhi-zhong, DING Jia-bo, MENG Shan-shan, YANG Han-chun.
Prokaryotic Expression of pp24 Gene of Md11 Strain of Marek’s Disease Virus .VIROLOGICA SINICA, 2004, 19(4)
: 369-372.
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摘要
通过PCR方法扩增MDV Md11株pp24基因的完整ORF，按正确的阅读框架将其克隆入原核表达载体pGEX-6P-1中，重组质粒转化BL21宿主菌后，经IPTG诱导表达。诱导菌体裂解物经SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）后，用山羊抗GST抗体进行Western-blotting试验，结果确证了GST-pp24融合蛋白的表达。将表达产物从凝胶中回收，免疫4周龄小白鼠，3次免疫后，采血分离血清。所得抗GST-pp24多克隆抗血清分别与GA株、Md11株和CVI988株MDV感染的鸡胚成纤维细胞（CEF）进行间接免疫荧光试验（IFA），结果表明大肠杆菌表达的pp24融合蛋白至少保留了部分天然pp24蛋白的抗原性。
Prokaryotic Expression of pp24 Gene of Md11 Strain of Marek’s Disease Virus
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1. Animal Science and Technology College, Shandong Agricultural University, Shandong, Taian 271018, China
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College of Veterinary Science, China Agricultural University, Beijing 100094, China
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Corresponding author:
CUI Zhi-zhong,
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Abstract
pp24 gene was amplified from genomic DNA of Marek’s disease viruses (MDV) Md11 strain by polymerase chain reaction(PCR) and then cloned into pGEX-6P-1 according to the right open reading frame(ORF). The recombinant plasmid was transformed into E.coli BL21 strain for expression with the induction of IPTG. SDS-PAGE and Westen-blotting with goat-anti-GST antibody validated the expression of GST-pp24 fusion protein. The expressed specific band was excised from the gel and injected into four-week-old mice 3 times, then the antiserum was collected from the mice and used for IFA with Chicken Embryonic Fibroblasts(CEF) infected by MDV Md11, CVI988 and GA strains respectively. The positive staining was found in the MDV plaques, which shows that the fusion protein of pp24 in vitro expressed has some epitopes of natural pp24.
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References
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Proportional views
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