Cloning of toxin and outer membrane protein genes in Actinobacillus pleuropneumoniae and immunity of the recombinant protein

• Main Authors: Tai-Sheng Chiang, 姜臺生
• Other Authors: Chao-Fu Chang
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/92173969095112108465

博士 === 國立臺灣大學 === 獸醫學研究所 === 89 === Cloning and expression of the outer membrane lipropteins（OmlA）of Actinobacillus pleuropneumoniae serotypes 1 and 5, transferrin binding proteins（Tbp2） of serotypes 1, 5 and 7, and A. pleruopneumoniae toxin (Apx) of serotype 5.The open reading frames were amplified from A. pleuropneumoniae chromosomal DNA by using PCR, and cloning into pCR-TOPO. Subsequently, all genes were subscloned into expression vectors pKK223-3, pGEX-4T-2, and pEXT20, to express either native form or fusion proteins. The product expressed by pKK223-3 vector was toxicity to E. coli. The GST-fusion proteins were well expressed OmlA of serotypes 1 and 5 and Tbp2 of serotype 7 did not show aggregate formation, while most of Tbp2 of serotypes 1 and 5 were insoluble aggregates as inclusion body. The pKK223-3 that contained ApxI and ApxII were expressed well, while the product of pEXT20 that contained ApxIII was poor. The recovered porcine serum of serotype 5 probed with Tbp2 and OmlA of serotype 5 showed strongly reaction that analyzed by immunoblot, while was reacted with moderately against OmlA of serotype 1, and weakly against Tbp2 of serotypes 1 and 7. All of proteins were stable that stored at －20 ℃ for 3 months of. The efficacy of recombinant vaccine of ApxI and ApxII was showed the protection in lung of the immunized porcine, and better than that of commercial bacterin.