GP5蛋白诱导表达及其多克隆抗体制备与PRRS和TGE共感染检测

发布时间：2018-06-05 11:29

本文选题：PRRSV + GP5蛋白　；参考：《西北农林科技大学》2015年硕士论文

【摘要】：利用本课题组前期构建的pET-28a-GP5-BL21,诱导表达并纯化PRRSV GP5蛋白,用获得的纯化的PRRSV GP5免疫小鼠,制备针对GP5的多克隆抗体,为GP5蛋白的检测提供材料。同时以基于纯化的GP5建立的GP5抗体检测的ELISA方法和商品化猪传染性胃肠炎病毒(TGEV)抗体ELISA检测试剂盒对采集自陕西省部分地市的猪血清进行检测,为陕西省猪群的PRRS和TGE进行血清学调查,以期为陕西省猪场猪繁殖与呼吸综合征和猪传染性胃肠炎的综合防控提供科学依据。1.PRRS GP5的诱导表达、纯化与鉴定将pET-28a-GP5-BL21接种于卡那抗性的液体培养基中,培养10-12h,过夜活化。然后按1:20接菌,37℃、220r/min,震荡培养至OD600nm约为0.6左右,取1mL菌液作为未诱导前的对照,其余加入终浓度为lmmol/L的IPTG诱导培养4h,取1mL菌液为样品。将样品处理后进行SDS-PAGE,确定目的蛋白是否表达。诱导后剩余菌液样品离心弃去上清液留沉淀,PBS重悬沉淀后超声波碎仪破碎,然后4℃、12000r/min离心分别收集上清和沉淀。用8mol/L的尿素重悬沉淀,样品处理后做SDS-PAGE确定蛋白的表达形式。之后进行大量表达,亲和层析法进行纯化,SDS-PAGE鉴定纯化的效果。Western blot鉴定的反应原性。结果诱导表达的GP5蛋白分子质量约为15ku,与预期的大小相符,表达的蛋白主要以包涵体形式存在,SDS-PAGE结果表明获得了纯化的目的蛋白;以PRRSV阳性猪血清进行Western blot检测,结果在约15ku处有印迹条带,表明表达的蛋白具有良好的反应原性。2.PRRS GP5的多克隆抗体的制备取纯化的GP5蛋白免疫6-8周龄昆明系小鼠,每只腹腔注射重组蛋白50μg,共免疫4次,每次间隔2周。其中第一次免疫使用弗氏完全佐剂,其他3次使用弗氏不完全佐剂。每次免疫当天割尾采血后进行免疫,最后一次免疫后第十天摘眼球采血,按常规方法分离血清-20℃备用。利用ELISA方法检测小鼠血清中的抗GP5蛋白的抗体效价。结果ELISA检测,制备的血清抗体效价均在1:10000以上,最高可达1:1024000以上。3.陕西省部分地市猪血清检测利用商品化的TGEV抗体ELISA检测试剂盒和实验室基于纯化的GP5建立的GP5抗体间接ELISA检测方法,对采集自陕西省6个地市的409份猪血清进行检测。结果采集的409份血清中GP5抗体阳性的有8份,TGEV抗体阳性的有65份,两者共阳性的有2份。本研究成功获得了诱导表达并纯化的PRRSV GP5蛋白,表达的蛋白主要以包涵体的形式存在,反应原性良好;通过免疫小鼠制备了抗GP5蛋白的多克隆抗体,血清抗体效价均在1:10000以上,最高达1:1024000以上;采集的409份血清中GP5抗体阳性的8份,TGEV抗体阳性的65份,两者共阳性的有2份,表明陕西省部分地市猪群中存在PRRSV与TGEV共感染现象。
[Abstract]:The PRRSV GP5 protein was induced and purified by pET-28a-GP5-BL21, which was constructed by the previous group, and the purified PRRSV GP5 was used to prepare the polyclonal antibody against GP5 and provide materials for the detection of GP5 protein. The ELISA method of GP5 anti physical examination based on the purified GP5 and the commercialized infectious gastroenteritis of swine was also used. TGEV antibody ELISA detection kit was used to detect the serum of pigs collected from partial city in Shaanxi province. The serological investigation was carried out for the PRRS and TGE of the pigs in Shaanxi Province, in order to provide the comprehensive prevention and control of porcine reproductive and respiratory syndrome and swine infectious gastroenteritis in Shaanxi province. The purification and identification will be based on the expression of.1.PRRS GP5. PET-28a-GP5-BL21 was inoculated in the liquid culture medium of kanamycin resistance. 10-12h was cultured and activated overnight. Then at 1:20, 37 degrees, 220r/min, concussion was cultured to about 0.6. The 1mL bacteria liquid was taken as the uninduced control. The rest was induced by IPTG in the final concentration of lmmol/L, and the 1mL bacteria solution was taken as the sample. The samples were treated for SD, and SD was processed for SD. SD S-PAGE, determine whether the target protein is expressed or not. After induction, the residual liquid samples are centrifuged and removed to the supernatant and deposited, and PBS is suspended by ultrasonic breakage after heavy suspension. Then 4 degrees centigrade, 12000r/min centrifugation is used to collect the supernatant and precipitate respectively. The expression of the protein is determined by SDS-PAGE in 8mol/L. After the sample is treated, the expression of the protein is determined. Then a large number of tables are made. It was purified by affinity chromatography and SDS-PAGE was purified by.Western blot. The results showed that the expression of GP5 protein was about 15ku, in accordance with the expected size, the protein expressed mainly in the form of inclusion body, and the result of SDS-PAGE showed that the purified target protein was obtained; PRRSV positive pig blood was cleared into the protein. The results of Western blot detection showed that there was an imprinted strip at about 15ku, indicating that the expressed protein had a good reactivity of the polyclonal antibody of.2.PRRS GP5 for the preparation of the purified GP5 protein for the immunization of 6-8 weeks old Kunming mice, each intraperitoneal injection of recombinant protein 50 mu g, immunized for 4 times, each interval of 2 weeks. All the adjuvant, the other 3 use of Freund incomplete adjuvant. Immunization at the end of the day after each immunization. After the last immunization, the blood was picked up tenth days after the final immunization. The serum -20 centigrade was separated by the routine method. The antibody titer of the anti GP5 protein in the serum of mice was detected by ELISA. Results the serum antibody titer was measured by ELISA. Above 1:10000, the pig serum of up to 1:1024000 above.3. in Shaanxi province was detected by commercial TGEV antibody ELISA detection kit and the GP5 antibody indirect ELISA detection method based on purified GP5 in laboratory, and 409 pig blood samples collected from 6 cities in Shaanxi province were detected. The results collected 409 sera GP5 There were 8 positive antibodies and 65 TGEV positive antibodies in 2 copies. The study successfully obtained the PRRSV GP5 protein, which was induced and purified. The protein expressed mainly in the form of inclusion body, and the reactivity was good. The polyclonal antibody of anti GP5 egg white was prepared by immunizing mice, and the titer of serum antibody was in 1:10000. Above, up to 1:1024000 above 1:1024000; 8 of the 409 sera were positive for GP5, 65 of TGEV antibody positive, and 2 were positive, indicating that there was a co infection between PRRSV and TGEV in some local pigs in Shaanxi province.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28

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