摘要
目的原核表达并纯化麻疹病毒(measles virus,MV)核蛋白(N),免疫家兔后制备兔抗MV N抗血清,并鉴定其特异性。方法以MV-S191疫苗株基因组为模板,PCR扩增N基因3个片段,构建重组表达质粒pGEX-MV-N1、pGEXMV-N2和pGEX-MV-N3,分别转化至E.coli BL21(DE3)中诱导表达N-末端带有GST标签的融合蛋白GST-MV-N1、GST-MV-N2和GST-MV-N3,纯化后,分别与等体积弗氏完全佐剂(初次免疫)、弗氏不完全佐剂(加强免疫)混合,乳化均匀后各免疫1只雌性家兔,共免疫6次,采集全血,分离血清获得兔抗MV N抗血清,Western blot法及间接免疫荧光试验(indirect immunofluorescence assay,IFA)鉴定其特异性。结果重组表达质粒pGEX-MV-N1、pGEX-MV-N2和pGEX-MV-N3经双酶切、PCR及测序鉴定证明构建正确;表达的GST-MV-N1和GST-MV-N3融合蛋白相对分子质量分别约42000和39000,主要以包涵体形式存在,纯度分别为96%和85%,而上清和沉淀中均未见GST-MV-N2融合蛋白的表达;兔抗MV N3抗血清能特异性识别MV-S191感染细胞后表达的N蛋白,且特异性高于兔抗MV-N1抗血清。结论成功制备了兔抗MV N抗血清,为重组MV疫苗的研发以及MV结构蛋白抗体的制备提供了技术支持。
Objective To express and purify the N proteins of measles virus(MV)in prokaryotic cells,immunize rabbits to prepare rabbit antisera against MV N proteins,and identify the specificity.Methods Using the genome of MV-S191vaccine strain as template,three fragments of N gene were amplified by PCR to construct the recombinant expression plasmids pGEX-MV-N1,pGEX-MV-N2 and pGEX-MV-N3,which were then transformed into E.coli BL21(DE3)and induced to express the fusion proteins GST-MV-N1,GST-MV-N2 and GST-MV-N3 with GST tags on the N-terminal,respectively.After purification,the fusion proteins were mixed with equal volumes of Freund's complete adjuvant(for initial immunization)and Freund's incomplete adjuvant(for booster immunization),separately,and were used to immunize female rabbits for six times after evenly emulsification,with one rabbit for each protein.The whole blood was collected to isolate the rabbit antisera against MV N proteins,and the specificity of rabbit antisera was identified by Western blot and indirect immunofluorescence assay(IFA).Results The recombinant expression plasmids pGEX-MV-N1,pGEX-MV-N2 and pGEXMV-N3 were constructed correctly as identified by double enzyme digestion,PCR and sequencing.The fusion proteins GSTMV-N1 and GST-MV-N3 mainly existed in the form of inclusion bodies,with a relative molecular mass of about 42000 and39000 as well as a purity of 96%and 85%,respectively.However,GST-MV-N2 fusion protein was not detected in the supernatant and precipitate.The rabbit anti-MV-N3 antisera specifically recognized the N protein expressed by MV-S191infected cells,and the specificity was higher than that of rabbit anti-MV-N1 antisera.Conclusion In this study,rabbit antiMV-N antisera was successfully prepared,which provides technical support for the development of recombinant MV vaccine and the preparation of MV structural protein antibody.
作者
杨志辉
安欢欢
吴杰
孟胜利
郭靖
申硕
YANG Zhihui;AN Huanhuan;WU Jie;MENG Shengli;GUO Jing;SHEN Shuo(Research Division I of Viral Vaccines,Wuhan Institute of Biological Products Co.,Ltd.,Wuhan 430207,Hubei Province,China)
出处
《中国生物制品学杂志》
CAS
CSCD
2024年第9期1025-1029,1036,共6页
Chinese Journal of Biologicals
基金
湖北省技术创新专项(2017ACA078)。
关键词
麻疹病毒
核蛋白
兔抗血清
原核表达
纯化
Measles virus(MV)
N protein
Rabbit antisera
Prokaryotic expression
Purification
