Basagoudanavar S H, Goswami P P, Tiwari V, Pandey A K, Singh N
National Biotechnology Centre, Indian Veterinary Research Institute, Izatnagar 243122, India.
Vet Res Commun. 2004 Apr;28(3):209-24. doi: 10.1023/b:verc.0000017371.68083.04.
The full-length open reading frame coding for a potentially immunogenic 35 kDa protein of Mycobacterium avium paratuberculosis was generated using polymerase chain reaction technology. The gene was inserted in-frame into Escherichia coli expression plasmid pQE32. The resulting recombinant plasmid pPMP35 was transformed into E. coli M15. Analysis of the E. coli induced with isopropyl-beta-D-thiogalactopyranoside revealed that the protein accumulated into the cytoplasm as insoluble inclusion bodies. The level of expression of the recombinant 35 kDa protein (P35) was more than 30% of the total protein of E. coli cells. Expression of the recombinant protein was confirmed by immunoblotting. The P35 reacted with a rabbit antiserum raised against a sonicate of M. a. paratuberculosis. The protein was also recognized by serum from a goat with clinical paratuberculosis. Further, a polyclonal antiserum against P35 recognized a 35 kDa band in a membrane fraction of M. a. paratuberculosis. Also, the protein provoked a significant skin reaction in outbred guinea pigs sensitized with M. a. paratuberculosis, as well as in those sensitized with Mycobacterium avium. The results indicate that the 35 kDa protein of M. a. paratuberculosis is a membrane protein, having a role in the cellular immune response.
利用聚合酶链反应技术生成了编码鸟分枝杆菌副结核亚种一种潜在免疫原性35 kDa蛋白的全长开放阅读框。该基因以读码框方式插入大肠杆菌表达质粒pQE32。将所得重组质粒pPMP35转化到大肠杆菌M15中。用异丙基-β-D-硫代半乳糖苷诱导大肠杆菌后分析发现，该蛋白以不溶性包涵体形式积累在细胞质中。重组35 kDa蛋白（P35）的表达水平超过大肠杆菌细胞总蛋白的30%。通过免疫印迹法证实了重组蛋白的表达。P35与针对鸟分枝杆菌副结核亚种超声裂解物产生的兔抗血清发生反应。该蛋白也被患有临床副结核病的山羊血清识别。此外，针对P35的多克隆抗血清在鸟分枝杆菌副结核亚种的膜组分中识别出一条35 kDa的条带。而且，该蛋白在用鸟分枝杆菌副结核亚种致敏的远交豚鼠以及用鸟分枝杆菌致敏的豚鼠中引发了显著的皮肤反应。结果表明，鸟分枝杆菌副结核亚种的35 kDa蛋白是一种膜蛋白，在细胞免疫反应中起作用。
