文献类型：期刊文献

中文题名：猪肺炎支原体P97基因重组腺病毒的构建及其免疫原性

英文题名：Construction and immunogenicity of the recombinant adenovirus containing P97 gene of Mycoplasma hyopneumoniae

作者：王凯[1];杨青春[1];陈绍红[2];刘艳芬[1];刘铀[2]

机构：[1]广东海洋大学农学院,广东湛江524088;[2]广东海洋大学现代生化中心,广东湛江524088

年份：2015

卷号：45

期号：11

起止页码：1114

中文期刊名：中国兽医科学

外文期刊名：Chinese Veterinary Science

收录：CSTPCD、、北大核心2014、CSCD2015_2016、北大核心、CSCD

基金：广东省科技计划项目(0809035)

语种：中文

中文关键词：猪肺炎支原体;黏附因子;重组腺病毒载体;疫苗

外文关键词：Mycoplasma hyopneumoniae; adherence factor; recombinant adenovirus vector; vaccine;

中文摘要：采用PCR扩增了猪肺炎支原体(Mhp)R659株黏附因子P97基因,测定了其核苷酸序列,并将其克隆至穿梭质粒pacAd5CMVK-NpA上。在脂质体介导下将PacⅠ酶线性化的重组穿梭质粒pacAd-P97和腺病毒骨架质粒pacAd5 9.2-100共转染AD-293细胞,包装出重组腺病毒Ad-P97,用RT-PCR、Westernblot和间接免疫荧光试验检测重组P97蛋白在AD-293和PK15细胞中的表达。之后,用Ad-P97抗血清检测Mhp对宿主细胞的黏附作用以及Ad-P97在仔猪体内的免疫应答反应。结果显示,P97基因由3 339个核苷酸组成,共编码1 113个氨基酸,其核苷酸序列高度保守;重组P97蛋白可在Ad-P97感染的AD-293细胞和PK15细胞中高效表达,但Ad-P97不能在PK15细胞中增殖并引起细胞病变;Ad-P97具有良好的免疫原性,能抑制Mhp对PK15细胞的黏附作用,诱导免疫仔猪产生Mhp凝集抗体。上述结果为进一步研制安全、高效的Mhp重组疫苗奠定了基础。

外文摘要：The P97 gene of the Mycoplasma hyopneumoniae（Mhp）standard strain R659 was amplified by PCR and the nucleotide sequence was sequenced,then the P97 gene was cloned into the pacAd5 CMVKNpA shuttle vector,and the AD-293 cells were co-transfected with linearized recombinant shuttle vector digested by restriction endonuclease PacⅠ and adenovirus backbone plasmid pacAd5 9.2-100.The recombinant adenovirus AD-P97 was expected to be packaged in the AD293 cells.The expression of recombinant P97 protein in AD-293 and PK15cells was detected by RT-PCR,Western-blot and indirect immunofluorescence,respectively.Finally,the inhibiting effect of Ad-P97 antiserum on Mhpadhesion to PK15 host cells and the serum agglutinating antibody level of the AD-P97 immunized piglets were used to evaluate the immunogenicity of the recombinant adenovirus Ad-P97.The results showed that the completed P97 gene of R659 strain,which was highly conserved,was consists of 3 339 nucleotides encoding 1 113 amino acids.The recombinant P97 protein can be efficiently expressed in Ad-P97 infected AD-293 and PK15cells,but the multiplication of recombinant virus in the Ad-P97 infected PK15cells and the cytopathic effect（CPE）was not observed.The Ad-P97 exhibited perfect immunogenicity,and was able to inhibit the Mphadhesion to PK15 cells,and induce agglutinating antibody against Mhpin immunized piglets.The above-mentioned results showed good prospects of developing a safe and effective recombinant Mhpvaccine.