摘要
利用DNAStar Protean功能筛选AP1和AP2主要抗原结构域,引入15位柔性多肽,通过重叠延伸PCR技术串联融合AP1、AP2主要抗原域基因,PCR扩增串联体基因片段,将其克隆至pET-30a（＋）载体,转化BL21（DM3）细胞,进行高效表达,目的蛋白经亲和层析纯化后进行Western blot鉴定。PCR扩增出320bp的AP1和759bp的AP2主要抗原域,经重叠延伸PCR获得了1 065bp的AP1、AP2串联体基因片段,DNA测序结果表明,无碱基的缺失、突变和移码。构建的重组表达载体经诱导能够可溶性表达,目的蛋白相对分子质量大小约46 000,纯化后重组蛋白的纯度〉96%,Western blot分析重组融合蛋白结果表明,能够被兔源抗无乳链球菌阳性血清所识别。重叠延伸PCR获得了AP1、AP2主要抗原域串联体,构建了pET-30a（＋）/AP1＋AP2重组表达载体,诱导目的蛋白呈可溶性表达,表达产物具有良好的免疫反应原性,为进一步研究基于AP1、AP2蛋白的致病机制,检测制剂及疫苗奠定了试验基础。
To express the main antigenic domain of the AP1, Inner Mongoka and AP2 ancillary protein in pilus insland of Streptococcus agalactiae, determine its antigenicity by the construction expression recombinant vector. Protean DNAStar was used to screen AP1 and AP2 major antigenic domains,and 15 flexible peptides were introduced in order to ensure the antigen,and the AP1 and AP2 genes were fused by overlapping extension PCR technique. The gene fragment was amplified,and cloned it to pET-30a（＋） vector,and transformed to BL21 （DM3） cells,in order to the efficient protein expression. The target protein was purifed by affinity chromatography and identified by Western blot. The AP1 of 759 bp and AP2 of 320 bp major antigenic domain were identified by protean screening. The CIS gene fragment obtained AP1 and AP2 series was 1 065 bp. DNA sequencing result showed that there was no base deletion, mutation and shift. The recombinant ex- pression vector was induced to express the soluble expression,the target protein was about 46 000, and the purity of the purified recombinant protein was above 90%. Western blot analysis showed that the recombinant protein can be identified by the positive serum of Streptococcus agalactiae. AP1 and AP2 major antigenic domains were obtained by overlap extension PCR. pET-30a-AP1＋ AP2 recombinant expression vector was constructed to induce the expression of the target protein, and the product had good antigen reactivity. This study provided an experimenal basis for further research on the immune vaccine based on AP1 and AP2 protein.
作者
布日额
王金良
吴金花
孙立杰
锡林高娃
董林
BU Ri-e1,2,3 , WANG Jin-liang4 , WU Jin-hua1,2,3 , SUN Li-jie1,2,3 , XILIN gao-wa1,2,3 , DONG Lin4(1. College of Life Science, Inner Mongolia University for Nationalities, Tongliao, Inner Mongolia 028043, China ; 2. Inner Mongolia Autonomous Region Engineering Technology Research Center of Prevention and Control the Pathogenic Bacteria in Milk, Tongliao, Inner Mongolia 028043, China ; 3. Research Institute for Pathogenic in Milk of Inner Mongolia University for Nationalities, Tongliao , Inner Mongolia 028043, China ; 4. Shandong Binzhou Animal Science and Veterinary Medicine Academy, Binzhou, Shandong 256600, Chin)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2018年第3期515-520,543,共7页
Chinese Journal of Veterinary Science
基金
国家自然科学基金资助项目(31560689,31760725)
内蒙古自治区乳源性致病菌防控工程技术研究中心开放课题资助项目(MDK2017016
MDK2017017)
内蒙古自治区“草原英才”创新创业团队人才计划资助项目(2017)
内蒙古科技厅社会发展领域计划资助项目(2017)
