减毒沙门菌介导防御素-3乳腺表达及对乳腺炎主要病原菌的抗菌效果

探讨减毒沙门菌介导的人HNP-3原前肽和前肽基因乳腺表达及其表达产物对牛乳腺炎主要病原菌的抗菌效果。构建人HNP-3原前肽和前肽基因的乳腺表达载体pEGFP-WAP-prepro-HNP-3和pEGFP-WAP-pro-HNP-3,电转化导入减毒沙门菌ΔcrpC78-1,构建重组菌ΔcrpC78-1（pEGFP-WAP-prepro-HNP-3）和ΔcrpC78-1（pEGFPWAP-pro-HNP-3）,重组菌分点注射到怀孕后20~22d母兔腹部乳腺分布区,收集兔分娩后不同时间的泌乳,Western-blot和荧光检测乳中HNP-3融合蛋白表达情况,ELISA检测乳中HNP-3融合蛋白的表达水平,常规方法分离乳腺炎主要病原菌,活菌计数法检测泌乳中HNP-3融合蛋白抑菌效果。结果显示,乳汁中分别在40 000和35 000处有prepro-HNP-3和pro-HNP-3融合蛋白阳性杂交信号,且在488nm处有黄绿色荧光;母兔在分娩后第5dHNP-3融合蛋白表达量最高,分别可达584μg/L和470μg/L,及至第25天表达量仍可达到277μg/L和213μg/L。活菌计数法结果表明分娩后第5d含prepro-HNP-3融合蛋白兔乳对牛乳腺炎主要病原菌大肠埃希菌、溶血巴氏杆菌、无乳链球菌、乳房链球菌、金黄色葡萄球菌和表皮葡萄球菌的抑菌率分别为92.0%、54.8%、46.4%、48.8%、86.9%和74.9%;含pro-HNP-3融合蛋白乳的抑菌率分别为84.9%、46.5%、40.6%、40.4%、71.1%和58.4%。结果表明,减毒沙门菌能够介导人HNP-3融合蛋白基因在乳腺上皮细胞中定位表达,表达的preproHNP-3和pro-HNP-3具有较强的生物学活性,对牛乳腺炎主要病原菌具有明显的抑制作用,为乳腺炎的生物防控及生物源性抗菌肽的研究提供了参考。

Expression of recombinant HNP-3 gene in mammary gland delivered by attenuated Salmonella choleraesuis and the detection of its antibacterial activity for mastitis pathogenic bacteria

To evaluate the expression of HNP-3prepro-peptide（prepro-HNP-3）and pro-peptide（pro-HNP-3）gene in mammary gland mediated by attenuated Salmonella choleraesuis and test the antibacterial activity for mammitis pathogenic bacteria.The specific mammary gland expression vector pEGFP-WAP-prepro-HNP-3and pEGFP-WAP-pro-HNP-3were transformed by electroporation into attenuated Salmonella ΔcrpC78-1and the recombinant bacteria ΔcrpC78-1（pEGFP-WAP-prepro-HNP-3）andΔcrpC78-1（pEGFP-WAP-pro-HNP-3）were constructed,the recombinant strains are injected into different hypodermic sites of rabbit mammary glands after pregnant 20-22d,collected the lactation after parturition in different times.Western-blot and fluorescence microscopy as well as ELISA analysis the fusion protein of prepro-HNP-3and pro-HNP-3expression in milk.Viable count analysis the antibacterial activity of the fusion proteins.The results showed that the milk obtained from rabbit contains biologically activity of the fusion protein of prepro-HNP-3and pro-HNP-3,and concentration were calculated to be about 584μg/L and 470μg/L on 5th day after parturition,at the 25d,the expression level of the fusion protein of preproHNP-3and pro-HNP-3remain to 277μg/L and 213μg/L.The results of the viable count showed that the fusion protein of prepro-HNP-3and pro-HNP-3expressed in rabbit milk have antibacterial activity on E.coli,Haemolysis papanicolaou bacillus,Streptococcus agalactiae,Streptococcus uberis,Staphylococcus aureus and Staphylococcus epidermidis,the fusion protein of preproHNP-3inhibition rates were 92.0%,54.8%,46.4%,48.8%,86.9%and 74.9%;the fusion protein of pro-HNP-3were 84.9%,46.5%,40.6%,40.4%,71.1% and 58.4%,respectively.The specific expression vector pEGFP-WAP-prepro-HNP-3and pEGFP-WAP-pro-HNP-3could be expressed in mammary gland mediated by attenuated Salmonella and the expressed product could secrete into milk and would be of biological activity.This study provides the useful information for preventing animal mastitis.