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  本文選題：PRRSV + GP5蛋白��； 參考：《西北農(nóng)林科技大學(xué)》2015年碩士論文

【摘要】：利用本課題組前期構(gòu)建的pET-28a-GP5-BL21,誘導(dǎo)表達并純化PRRSV GP5蛋白,用獲得的純化的PRRSV GP5免疫小鼠,制備針對GP5的多克隆抗體,為GP5蛋白的檢測提供材料。同時以基于純化的GP5建立的GP5抗體檢測的ELISA方法和商品化豬傳染性胃腸炎病毒(TGEV)抗體ELISA檢測試劑盒對采集自陜西省部分地市的豬血清進行檢測,為陜西省豬群的PRRS和TGE進行血清學(xué)調(diào)查,以期為陜西省豬場豬繁殖與呼吸綜合征和豬傳染性胃腸炎的綜合防控提供科學(xué)依據(jù)。1.PRRS GP5的誘導(dǎo)表達、純化與鑒定將pET-28a-GP5-BL21接種于卡那抗性的液體培養(yǎng)基中,培養(yǎng)10-12h,過夜活化。然后按1:20接菌,37℃、220r/min,震蕩培養(yǎng)至OD600nm約為0.6左右,取1mL菌液作為未誘導(dǎo)前的對照,其余加入終濃度為lmmol/L的IPTG誘導(dǎo)培養(yǎng)4h,取1mL菌液為樣品。將樣品處理后進行SDS-PAGE,確定目的蛋白是否表達。誘導(dǎo)后剩余菌液樣品離心棄去上清液留沉淀,PBS重懸沉淀后超聲波碎儀破碎,然后4℃、12000r/min離心分別收集上清和沉淀。用8mol/L的尿素重懸沉淀,樣品處理后做SDS-PAGE確定蛋白的表達形式。之后進行大量表達,親和層析法進行純化,SDS-PAGE鑒定純化的效果。Western blot鑒定的反應(yīng)原性。結(jié)果誘導(dǎo)表達的GP5蛋白分子質(zhì)量約為15ku,與預(yù)期的大小相符,表達的蛋白主要以包涵體形式存在,SDS-PAGE結(jié)果表明獲得了純化的目的蛋白;以PRRSV陽性豬血清進行Western blot檢測,結(jié)果在約15ku處有印跡條帶,表明表達的蛋白具有良好的反應(yīng)原性。2.PRRS GP5的多克隆抗體的制備取純化的GP5蛋白免疫6-8周齡昆明系小鼠,每只腹腔注射重組蛋白50μg,共免疫4次,每次間隔2周。其中第一次免疫使用弗氏完全佐劑,其他3次使用弗氏不完全佐劑。每次免疫當(dāng)天割尾采血后進行免疫,最后一次免疫后第十天摘眼球采血,按常規(guī)方法分離血清-20℃?zhèn)溆�。利用ELISA方法檢測小鼠血清中的抗GP5蛋白的抗體效價。結(jié)果ELISA檢測,制備的血清抗體效價均在1:10000以上,最高可達1:1024000以上。3.陜西省部分地市豬血清檢測利用商品化的TGEV抗體ELISA檢測試劑盒和實驗室基于純化的GP5建立的GP5抗體間接ELISA檢測方法,對采集自陜西省6個地市的409份豬血清進行檢測。結(jié)果采集的409份血清中GP5抗體陽性的有8份,TGEV抗體陽性的有65份,兩者共陽性的有2份。本研究成功獲得了誘導(dǎo)表達并純化的PRRSV GP5蛋白,表達的蛋白主要以包涵體的形式存在,反應(yīng)原性良好;通過免疫小鼠制備了抗GP5蛋白的多克隆抗體,血清抗體效價均在1:10000以上,最高達1:1024000以上;采集的409份血清中GP5抗體陽性的8份,TGEV抗體陽性的65份,兩者共陽性的有2份,表明陜西省部分地市豬群中存在PRRSV與TGEV共感染現(xiàn)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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S858.28

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本文編號：1981830