【摘要】：【目的】建立基于N與GP5蛋白抗原表位串聯(lián)的豬繁殖與呼吸綜合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)抗體ELISA檢測(cè)方法,為開發(fā)準(zhǔn)確、廉價(jià)的PRRSV抗體檢測(cè)試劑盒奠定基礎(chǔ)。【方法】將PRRSV的N蛋白和GP5蛋白抗原表位串聯(lián)優(yōu)化后進(jìn)行原核表達(dá),以表達(dá)的目的蛋白為抗原,通過(guò)優(yōu)化反應(yīng)條件建立檢測(cè)PRRSV抗體的ELISA方法,對(duì)其特異性、重復(fù)性進(jìn)行檢測(cè)。用建立的間接ELISA方法和商品化IDEXX試劑盒同時(shí)對(duì)臨床送檢的45份豬血清樣品進(jìn)行檢測(cè),計(jì)算其符合率。【結(jié)果】SDS-PAGE和Western Blot分析表明,表達(dá)的目的蛋白分子質(zhì)量約為24.7ku,具有良好的生物學(xué)活性,且為可溶性表達(dá)。目的蛋白經(jīng)純化后作為抗原包被ELISA平板,建立檢測(cè)PRRSV抗體的間接ELISA方法,優(yōu)化后的ELISA條件為:抗原(純化后的目的蛋白)包被量為5μg/mL、一抗(豬血清)1∶80稀釋、酶標(biāo)二抗1∶5 000稀釋,以含50g/L脫脂奶粉的PBST作為封閉液,37℃孵育60min,抗體(一抗和酶標(biāo)二抗)于37℃孵育90min,TMB避光顯色8min;間接ELISA的判定標(biāo)準(zhǔn)為:OD450≥0.345 2為陽(yáng)性,OD4500.345 2為陰性。所建立的間接ELISA方法特異性強(qiáng),對(duì)豬常見(jiàn)病原,如豬偽狂犬病毒、豬圓環(huán)病毒2型、豬瘟病毒和豬流行性乙型腦炎病毒高免血清檢測(cè)均為陰性;重復(fù)性好,組內(nèi)變異系數(shù)和組間變異系數(shù)分別為1.02%~3.94%和1.38%~4.83%。用所建立的間接ELISA方法對(duì)臨床送檢45份豬血清樣品的檢測(cè)陽(yáng)性率為84.44%(38/45),商品化IDEXX試劑盒檢測(cè)的陽(yáng)性率為80%(36/45),2種方法的符合率為94.73%(36/38)�！窘Y(jié)論】成功建立了基于N與GP5蛋白抗原表位串聯(lián)的PRRSV抗體ELISA檢測(cè)方法。
[Abstract]:[objective] to establish a ELISA method for detection of porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus,PRRSV) antibody based on N and GP5 epitopes. The cheap PRRSV antibody detection kit laid the foundation. [methods] the N protein of PRRSV and the antigen epitope of GP5 protein were optimized for prokaryotic expression, and the expressed target protein was used as antigen. A ELISA method for detection of PRRSV antibody was established by optimizing the reaction conditions, and its specificity and repeatability were detected. The indirect ELISA method and commercial IDEXX kit were used to detect 45 pig serum samples for clinical examination, and the coincidence rate was calculated. [results] SDS-PAGE and Western Blot analysis showed that, The molecular weight of the expressed target protein is about 24.7ku. it has good biological activity and soluble expression. Objective protein was purified as antigen to coat ELISA plate, and an indirect ELISA method for detection of PRRSV antibody was established. The optimized ELISA conditions were as follows: the amount of antigen (purified target protein) was 5 渭 g / mL, and the first antibody (pig serum) was diluted at 1:80. The enzyme labeled second antibody was diluted at 1:5, PBST containing 50g/L skim milk powder was incubated at 37 鈩，

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