重组CRM_(197)-4GnRH去势疫苗抗原分子的原核表达及免疫效果评价  

Prokaryotic expression and immunological evaluation of recombinant CRM_(197)-4GnRH castration vaccine molecules

作　　者：高华义 刘堃 张璐 王永芳 任东兴 郭钰洁 武毅[4] 申雁冰[1] 付旭彬 王敏[1] GAO Huayi;LIU Kun;ZHANG Lu;WANG Yongfang;REN Dongxing;GUO Yujie;WU Yi;SHEN Yanbing;FU Xubin;WANG Min(College of Biotechnology,Tianjin University of Science and Technology,Tianjin 300457,China;Tianjin Ringpu Bio-technology Co.,Ltd,Tianjin 300308,China;Tianjin Customs Animal and Plant and Foodstuffs Inspection Center,Tianjin 300461,China;College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]天津瑞普生物技术股份有限公司,天津300308 [3]天津海关动植物与食品检测中心,天津300461 [4]南京农业大学动物医学院,江苏南京210095

出　　处：《畜牧与兽医》2024年第3期69-76,共8页Animal Husbandry & Veterinary Medicine

基　　金：国家科学技术部资助项目(2014DFA31180);天津市科技支撑重点项目(20YFZCSN00120)。

摘　　要：通过大肠杆菌表达系统对CRM_(197)-4GnRH重组去势疫苗抗原进行表达,采用不同种类佐剂制备疫苗,研究其对大鼠的免疫去势作用,为获得1种免疫效果好、有效期长、生产成本低的促性腺激素释放激素(GnRH)免疫去势基因工程疫苗提供借鉴。采用白喉毒素无毒突变体(CRM_(197))与4拷贝GnRH串联,将CRM_(197)-4GnRH基因与表达载体连接,并通过优化试验确定大肠杆菌的最优表达条件,用SDS-PAGE和Western blot鉴定目标蛋白的表达情况。将融合蛋白与不同佐剂乳化,制备成9种测试疫苗后分别免疫大鼠,测定血清中GnRH抗体滴度和睾酮浓度。结果显示,重组大肠杆菌的最优表达条件为诱导时间表达5 h,培养基为LB液体培养基,IPTG诱导浓度为0.5 mmol/L;通过SDS-PAGE和Western blot试验,表明目标融合蛋白已成功表达;动物试验结果表明,重组疫苗水包油包水佐剂组、重组疫苗油包水佐剂组、GnRH+Th抗原(辅助型T细胞抗原)表位(G1抗原)油包水佐剂组测试疫苗免疫大鼠较其他组GnRH抗体滴度显著升高、睾酮浓度显著下降(P<0.05),表明这3种疫苗具有良好的免疫去势作用。研究结果为GnRH免疫去势疫苗的研制奠定了基础。The CRM_(197)-4GnRH antigen was expressed using an Escherichia coli expression system and vaccines were prepared with differ-ent adjuvants for immunization of rats to determine their immunocastration effects,aiming to provide insights for the development of a geneti-cally engineered GnRH-based vaccine with optimal immunogenicity,prolonged efficacy,and cost-effectiveness for immunocastration in ani-mal models.The CRM_(197)-4GnRH gene was linked to the expression vector using Diphtheria toxin CRM197(Corynebacterium Diphtheriae An-tigen,CRM_(197)-Conjugate)in series with four copies of GnRH(gonadotropin releasing hormone),and the optimal expression conditions of Escherichia coli were determined by optimization test.The expression of the target protein was identified by SDS-PAGE and Western blot.Nine vaccines utilized for testing purposes were prepared by emulsifying the fusion protein with different adjuvants,and were then immunized with GnRH antibody titers and testosterone concentration in the serum of rats.The results showed that the optimal expression conditions of Escherichia coli were determined as follows:induction time,5 h;LB liquid,medium;IPTG induction concentration,05 mmol/L.The tar-get fusion protein was successfully expressed through SDS-PAGE and Western blot analysis.The results of the animal experiments showed that the group immunized with the recombinant vaccine formulated with water-in-oil-in-water(W/O/W)adjuvant,the group immunized with the recombinant vaccine formulated with oil-in-water(O/W)adjuvant,and the group immunized with the GnRH+Th(Helper T cells)epitope(G1)formulated with oil-in-water(O/W)adjuvant,all exhibited a significant increase in GnRH antibody titer and a significant decrease in testosterone concentration,compared with the other groups(P<005).The present results indicate that the above three vaccines exhibited significant immunocastration effects,laying the foundation for the development of GnRH immunocastration vaccines.

关 键 词：CRM_(197) GNRH 大鼠 免疫去势 

分 类 号：S852[农业科学—基础兽医学]