【作者】 王艳梅；

【导师】 韩文瑜；

【作者基本信息】 吉林大学 ， 预防兽医学， 2019， 硕士

【摘要】 猪繁殖与呼吸综合征（Porcine Reproductive and Respiratory Syndrome PRRS）是由猪繁殖与呼吸病毒（Porcine Reproductive and Respiratory Syndrome Virus PRRSV）引起的一种高度传染性疾病。该病主要是以预防为主,疫苗接种是预防该病的首要措施。GP5蛋白是PRRSV重要的结构蛋白,存在六个重要的抗原决定簇,可以诱导中和抗体的产生,一直是疫苗研发的重要靶蛋白。中和性B细胞表位已经被确认为该病毒的中和性抗原区域。本研究旨在探究中和性B细胞表位诱导机体产生中和抗体及其作用。首先以合成中和性B细胞表位对7个猪场经不同商品化疫苗免疫的430份猪免疫临床血清进行检测,检测表明不同猪场的血清抗体水平阳性百分比在86.11%⁹8.15%之间。以中和性B细胞表位为目的展示序列,分别与靶向肽DC3pep、非靶向肽SCRMpep进行融合,通过噬菌体展示技术构建重组噬菌体M13-DC3pep-GP5-B和M13-SCRMpep-GP5-B,以临床PRRSV阳性血清进行western blot分析二者的免疫反应原性。以1.0×10¹³pfu/mL的重组噬菌体M13-DC3pep-GP5-B、M13-DC3pep-GP5-B混合弗氏佐剂和M13-SCRMpep-GP5-B免疫小鼠,对小鼠血清进行特异性抗体检测,表明重组噬菌体M13-DC3pep-GP5-B和M13-SCRMpep-GP5-B均具有良好的反应原性;M13-DC3pep-GP5-B混合弗氏佐剂免疫组的小鼠血清有特异性抗体产生,抗体水平在第四周达到最高为0.94;免疫组M13-DC3pep-GP5-B小鼠抗体水平在第六周达到最高为0.93,免疫组M13-SCRMpep-GP5-B的抗体水平在第六周达到最高为0.634,免疫组M13-DC3pep-GP5-B的特异性抗体水平均高于免疫组M13-SCRMpep-GP5-B,二者差异显著（p<0.05）。将重组噬菌体M13-DC3pep-GP5-B、M13-DC3pep-GP5-B混合氢氧化铝佐剂和M13-SCRMpep-GP5-B以1.0×10¹³pfu/mL的剂量免疫断奶仔猪,对仔猪血清进行特异性抗体检测,以临床分离PRRSV毒株对仔猪血清进行中和试验,表明M13-DC3pep-GP5-B混合氢氧化铝佐剂免疫组的抗体水平在末次免疫后第四周达到最高为0.687,且其抗体水平显著高于免疫M13-DC3pep-GP5-B组和M13-SCRMpep-GP5-B组（p<0.05）;免疫组M13-DC3pep-GP5-B的特异性抗体水平高于免疫组M13-SCRMpep-GP5-B,无显著差异（P>0.05）;但是诱导机体产生的中和抗体水平较低,免疫血清只在低稀释度时对PRRSV呈现中和活性。为了进一步发现并验证猪繁殖与呼吸障碍综合征病毒（PRRSV）保护性抗原GP5蛋白羧基端新的中和性表位,本研究运用生物信息学软件预测PRRSV GP5蛋白羧基端的抗原性,全基因合成GP5蛋白羧基端168-198位氨基酸的编码序列,构建了重组噬菌体M13-GP5168-198aa,以临床PRRSV阳性血清进行western blot分析重组噬菌体表面展示的GP5 168-198aa多肽的免疫反应原性。以1.0×10¹³pfu/mL重组噬菌体混合氢氧化铝佐剂制备的免疫抗原,按照灭活疫苗免疫程序肌肉注射免疫PRRSV抗体阴性断乳仔猪,以临床分离PRRSV毒株进行中和试验。结果显示:以临床阳性血清进行Western blot分析表明重组噬菌体表面展示的GP5168-198aa多肽具有免疫反应原性;免疫仔猪诱导仔猪产生的中和性抗体在第六周达到最高,中和滴度为1:10.08。综上,人工合成PRRSV GP5蛋白的中和性B细胞表位可用于临床猪血清PRRSV抗体的检测;运用噬菌体展示技术展示了中和性B细胞表位,免疫断奶仔猪,诱导机体产生的中和抗体水平较低,免疫血清只在低稀释度时对PRRSV呈现中和活性;生物学软件预测GP5蛋白羧基端的抗原表位进行展示,免疫断奶仔猪,可以诱导仔猪产生中和性抗体,仔猪免疫血清在细胞水平上对PRRSV呈现良好的中和作用。为PRRSV GP5新型多表位疫苗的开发奠定了基础。更多还原

【Abstract】 Porcine Reproductive and Respiratory Syndrome（PRRS）is a highly contagious disease which caused by Porcine reproductive and respiratory syndrome virus.So far,the disease has been mainly treated with preventive measures,and vaccination is the primary measure to prevent the disease.GP5 protein is an important structural protein of PRRSV,and there are six important antigenic determinants,which can induce the production of neutralizing antibodies,so it has been an important protein for vaccine development.Among them,the neutralizing B epitope has been confirmed as the neutralizing antigen region of the virus.This study aimed to investigate the neutralizing antibodies produced by neutralizing B cell epitopes and their effects.A total of 430 positive field serums from 7 farms vaccined with different commercial vaccines respectively were tested by epitope B based indirect ELISA.The results showed that the percentage of positive serum antibody levels in different farms ranged from 86.11%to 98.15%。Using the neutralizing epitope B epitope as the target sequence,fuse with the targeting peptide DC3pep and the non-targeting peptide SCRMpep respectively,then construct the recombinant phage M13-DC3pep-GP5-B and M13-SCRMpep-GP5 by phage display technology,use clinical positive serum for western blot analysis of the immunogenicity of both.Mice were immunized with1.0×10¹³pfu/mL recombinant phage M13-DC3pep-GP5-B,M13-DC3pep-GP5-B mixed Freund’s adjuvant and M13-SCRMpep-GP5,then specific antibodies were detected of mouse serum;The results showed that the both the recombinant phage M13-DC3pep-GP5-B and M13-SCRMpep-GP5-B have a good reactogenicity.The mice immunized of M13-DC3pep-GP5-B mixed Freund’s adjuvant produced specific antibody,antibody levels reached a maximum of 0.94 in the fourth week;In the immunized group of M13-DC3pep-GP5-B,the antibody level of mice reached a maximum of 0.93 in the sixth week,In the immunized group of M13-SCRMpep-GP5-B,the antibody level of mice reached a maximum of 0.634 in the sixth week.The specific antibody produced by recombinant phage M13-DC3pep-GP5-B was higher than immunized by M13-SCRMpep-GP5-B,and the difference of them was significant（p<0.05）.The weaned piglets were immunized with recombinant phage M13-DC3pep-GP5-B、M13-DC3pep-GP5-B mixed aluminum hydroxide adjuvant and M13-SCRMpep-GP5-B at a dose of 1.0×10¹³ pfu/mL.The specific antibodies of piglet serum were detected,the neutralization test of piglets were tested by using clinical isolation of PRRSV strains;The results showed that the antibody level of piglets immunized M13-DC3pep-GP5-B mixed aluminum hydroxide adjuvant reached the highest level of 0.687 in the fourth week after the last immunization,and its antibody level was significantly higher than piglets immunized M13-DC3pep-GP5-B group and M13-SCRMpep-GP5-B group（p<0.05）;the specific antibody level of piglets immunized by M13-DC3pep-GP5-B was higher than that piglets immunized by M13-SCRMpep-GP5-B,the difference was not significant（P>0.05）;The results showed that the neutralizing antibodies produced by piglets induced by M13-DC3pep-GP5-B and M13-SCRMpep-GP5-B were not sufficient to produce protective effects.However,the level of neutralizing antibodies is low,and the serum exhibits neutralizing activity against PRRSV only at low dilution.To find and validate the new neutralizing epitope at the carboxy terminal of protective antigen GP5 protein from porcine reproductive and respiratory syndrome virus（PRRSV）,in this study the antigenicity of the carboxy terminus of PRRSV GP5protein was predicted by bioinformatics software,the coding sequence of amino acid168-198 at the carboxy terminus of GP5 protein was synthesized,then the recombinant phage M13-GP5 168-198 was constructed.The immunoreactivity of the GP5 168-198 polypeptide displayed on the surface of the recombinant phage was analyzed by western blot using clinically positive serum.The immunized antigen,prepared by 1.0x10¹³ pfu/mL recombinant phage mixed with aluminum hydroxide adjuvant was injected intramuscularly into the PRRSV antibody negative weaned piglets according to inactivated vaccine immunization program,,serum was separated for neutralization test with the clinical isolated virulent strain of PRRSV;The results of western blot showed that the GP5 168-198 aa polypeptide displayed on the surface of the recombinant phage had immunoreactivity,and the neutralizing antibody induced by the immunized piglets reached the highest in the sixth week,and the neutralizing titer was reached at 1:10.08.In summary,synthetic neutralizing B cell epitopes of PRRSV GP5 protein can be used to detect PRRSV antibodies in clinical pig serum;The neutralizing B cell epitopes was display by phage display technology,By immunizing weaned piglets,the level of neutralizing antibodies is low,and the serum exhibits neutralizing activity against PRRSV only at low dilution;The epitope of the carboxy terminal of GP5 protein predicted by biological software was display by phage display technology,By immunizing weaned pigs,the piglets could produce neutralizing antibodies,piglet serum showed good neutralization at the cellular level,which laid the foundation for the development of a novel multi-epitope vaccine of PRRSV GP5.更多还原