猪瘟病毒C、NS5A、NS5B蛋白原核表达及应用
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摘要
本试验在大肠杆菌表达系统中得到了CSFV重组的C、NS5A和NS5B蛋白。通过Western blot试验证实3个蛋白都能与猪瘟阳性血清发生特异性反应,这为基因工程疫苗研究和建立特异性血清学诊断方法以及3个蛋白的相关功能性研究奠定了基础。
以纯化的重组C、NA5A、NS5B蛋白为诊断抗原,以pET-32a (+)空载体表达蛋白为阴性抗原同时包被96孔酶标板,建立检测CSFVC、NA5A、NS5B抗体的间接ELISA方法,并且进行了条件优化,与IDEXX公司猪瘟抗体ELISA试剂盒检测结果比较,重组C蛋白的一致性最高,为85.37%。
利用所建立的C-ELISA方法对302份不同年龄段(3日龄～免疫后16周龄)猪血清、以及母猪的血清进行检测,并且与IDEXX公司CSFV-Ab ELISA检测试剂盒检测结果进行了比较,母猪血清检测结果一致性较高,说明可以用C-ELISA检测母猪血清；仔猪血清的符合率低,与仔猪自身的免疫系统有关。通过两种ELISA方法的比较说明CSFV C抗体持续的时间不及E2,但检出的时间却早于CSFV E2抗体。
In the study,Recombinant proteins C、NS5A and NS5B of CSFV were expressed in E.coli expression system.The result of western blot showed the three proteins could be well identified by CSFV positive serum respectively exhibiting good antigenicity. The achievement of proteins C、NS5A and NS5B provides good preparation for genetically engineered vaccine and the establishment of specific serological diagnosis, and related function study of the three proteins.
Offered advanced technology for diagnosis of the disease of CSFV. Based on the purified recombinant proteins C、NS5A and NS5B as diagnosis antigens and the expression of empty vector as negative antigen, an indirect ELISA method for detection of CSFV antibodies was developed. And the conditions were optimized.Compared with CSFV-Ab test kit of IDEXX, the result showed:the best consistency is the C-ELISA.
Methods Serum specimens from 302 pigs of different ages from 3 days to 16 weeks old pigs and sows, were tested by the indirect ELISA for specific antibody against recombinant protein C. Comparing with CSFV-Ab test kit of IDEXX pointed that sows serum testing consistency is higher than piglets. It is showed we can use C-ELISA to test the serum of sows. Comparison of ELISA method in two shows the duration of CSFV C antibody is less than E2, but the detection of the time is earlier than E2.
以纯化的重组C、NA5A、NS5B蛋白为诊断抗原,以pET-32a (+)空载体表达蛋白为阴性抗原同时包被96孔酶标板,建立检测CSFVC、NA5A、NS5B抗体的间接ELISA方法,并且进行了条件优化,与IDEXX公司猪瘟抗体ELISA试剂盒检测结果比较,重组C蛋白的一致性最高,为85.37%。
利用所建立的C-ELISA方法对302份不同年龄段(3日龄～免疫后16周龄)猪血清、以及母猪的血清进行检测,并且与IDEXX公司CSFV-Ab ELISA检测试剂盒检测结果进行了比较,母猪血清检测结果一致性较高,说明可以用C-ELISA检测母猪血清；仔猪血清的符合率低,与仔猪自身的免疫系统有关。通过两种ELISA方法的比较说明CSFV C抗体持续的时间不及E2,但检出的时间却早于CSFV E2抗体。
In the study,Recombinant proteins C、NS5A and NS5B of CSFV were expressed in E.coli expression system.The result of western blot showed the three proteins could be well identified by CSFV positive serum respectively exhibiting good antigenicity. The achievement of proteins C、NS5A and NS5B provides good preparation for genetically engineered vaccine and the establishment of specific serological diagnosis, and related function study of the three proteins.
Offered advanced technology for diagnosis of the disease of CSFV. Based on the purified recombinant proteins C、NS5A and NS5B as diagnosis antigens and the expression of empty vector as negative antigen, an indirect ELISA method for detection of CSFV antibodies was developed. And the conditions were optimized.Compared with CSFV-Ab test kit of IDEXX, the result showed:the best consistency is the C-ELISA.
Methods Serum specimens from 302 pigs of different ages from 3 days to 16 weeks old pigs and sows, were tested by the indirect ELISA for specific antibody against recombinant protein C. Comparing with CSFV-Ab test kit of IDEXX pointed that sows serum testing consistency is higher than piglets. It is showed we can use C-ELISA to test the serum of sows. Comparison of ELISA method in two shows the duration of CSFV C antibody is less than E2, but the detection of the time is earlier than E2.
引文
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