摘要： 通过原核表达的方法得到血清4型禽腺病毒的主要结构蛋白六邻体蛋白；根据GenBank中血清4型禽腺病毒六邻体蛋白的基因序列，设计一对特异性引物，利用普通PCR方法扩增得到hexon基因的全长序列。将PCR扩增得到的血清4型禽腺病毒六邻体基因片段，在其两端插入酶切位点后克隆至载体pMD18T中，经PCR鉴定和测序分析正确后，与原核表达载体pET-32a进行连接，构建pET-32a-hexon原核表达载体，并对其进行双酶切和测序鉴定。将构建成功的表达载体转化至BL21（DE3）中，用1 mmol/L的IPTG进行诱导表达，对得到的重组蛋白进行SDS-PAGE分析和Western Blot鉴定；经双酶切鉴定和测序鉴定，pET-32a-hexon原核表达载体构建成功，插入的六邻体蛋白基因片段大小为2 814 bp，在1 mmol/L浓度IPTG诱导3 h时重组蛋白表达量最高，且重组蛋白主要以包涵体形式存在于沉淀中。SDS-PAGE分析结果显示，融合蛋白分子量为118 ku。免疫印迹分析结果显示，融合蛋白可被抗FAdV-4的血清和HIS标签抗体特异性识别；成功表达了血清4型禽腺病毒的主要结构蛋白六邻体蛋白，且得到的重组蛋白具有良好的反应原性，可用于血清4型禽腺病毒的进一步研究中，为血清4型禽腺病毒病的预防和治疗奠定了良好的基础。

关键词: 血清4型禽腺病毒, 六邻体蛋白, 原核表达

Abstract: To obtain the major structural protein hexon protein of serum type 4 avian adenovirus by prokaryotic expression; A pair of specific primers was designed according to the gene sequence of the hexon protein of Fowl adenovirus serotype 4 (FAdV-4) in GenBank. The full-length sequence of hexon gene was amplified by ordinary PCR. The serotype 4 avian adenovirus gene fragment obtained by PCR amplification was inserted into the vector pMD18T after inserting restriction enzyme sites at both ends of the gene. The PCR analysis and sequencing analysis showed that the gene was verified with the prokaryotic expression vector pET-32a. The constructed expression vector was transformed into BL21(DE3) and induced to express with 1 mmol/L IPTG. Analyzing the recombinant protein by SDS-PAGE and Western Blot; By the double-enzyme digestion and sequence analysis,the prokaryotic expression vector of pET-32a-hexon was successfully constructed. The size of the inserted hexon protein gene fragment was 2 814 bp. The recombinant protein expression was highest at 3 h after induction with 1 mmol/L IPTG,and the recombinant protein was mainly inclusion body forms exist in sediments. The SDS-PAGE analysis showed that the fusion protein had a molecular weight of 118 ku. Western Blot analysis showed that the fusion protein could be specifically recognized by anti-FAdV-4 serum and HIS tag antibody. This experiment successfully expressed the major structural protein hexon protein of serum type 4 avian adenovirus,and the fusion protein obtained had good reactogenicity,which could be used in the further research of serum type 4 avian adenovirus. It laid a good foundation for the prevention and treatment of serum type 4 fowl adenovirus disease.

Key words: Fowl adenovirus serotype 4(FAdV-4), Hexon protein, Prokaryotic expression

中图分类号: 

• Q78

• S432.4