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确定重组HBV表面抗原和HCV核心抗原对奶牛的最适免疫剂量、免疫次数及间隔时间,观察牛奶中抗HBV与抗HCV的效价与消长规律。表达HBV表面抗原和HCV核心抗原的重组菌经IPTG诱导后,目的蛋白以包涵体形式存在。放大发酵工艺,菌体发酵密度达40g/L,目的蛋白表达量为26%～30%。将包涵体变性、复性后,测定蛋白含量,并用SDS-PAGE鉴定。采用不同的剂量和不同的抗原处理方法免疫奶山羊和奶牛,检测HBV抗体、HCV抗体、干扰素与白介素等细胞因子。免疫奶山羊羊奶中的HBV抗体效价最高为1∶80,HCV抗体效价最高为1∶20;6个月后,HBV抗体效价无明显下降,HCV抗体效价下降明显。对奶牛5个批次的免疫结果显示,免疫次数应多于3次,免疫剂量以每头牛300μg为宜,间隔时间以1个月为宜。免疫牛奶中HBV抗体的阳性率约为66.0%,抗体效价最高为1∶160;HCV抗体阳性率约为17.0%,维持时间较短;孕牛比旺奶牛产生抗体的效价高。免疫牛奶中检测到了干扰素和白介素等细胞免疫活性因子。
To determine the optimal dose of immunization, the number of immunization and the interval time of recombinant HBV surface antigen and HCV core antigen in dairy cows to observe the titer and growth of anti-HBV and anti-HCV in milk. Recombinant bacteria expressing HBV surface antigen and HCV core antigen were induced by IPTG and the target protein existed as inclusion bodies. Amplification fermentation process, bacterial fermentation density of 40g / L, the target protein expression of 26% to 30%. The inclusion bodies were denatured and refolded, and the protein content was determined and identified by SDS-PAGE. Dairy cattle and goats were immunized with different doses and different antigen treatments to detect cytokines such as HBV antibodies, HCV antibodies, interferons and interleukins. The highest titers of HBV antibody in immunized goat and goat milk were 1:80, and the highest titers of HCV antibody were 1:20. After six months, the titers of HBV antibody did not decrease obviously, and the antibody titers of HCV antibody decreased obviously. Immunization of 5 batches of cows showed that the number of immunizations should be more than 3 times and that the dosage of immunization should be 300μg per cow. The interval should be 1 month. The positive rate of HBV antibody in immunized milk was about 66.0%, the highest antibody titer was 1:160, the positive rate of HCV antibody was about 17.0%, and the maintenance time was short. The antibody titer of pregnant cow than that of Wang cattle was high. Immune milk detected interferon and interleukin and other cellular immune activity factor.