爲建立一種(zhǒng)檢測非洲豬瘟病毒（ASFV）抗體劇開的間接ELISA（iELISA）方法，對(duì)構建的ASF放子V P30基因表達工程菌誘導表達後唱通(hòu)，將(jiāng)獲取的重組ASFV P答鐘30蛋白進(jìn)行純化和Western-blo文金t檢測，然後(hòu)以純化的重組蛋白爲抗原，建笑報立了ASFV抗體iELISA檢測方法，并進(jì你可n)行了特異性、靈敏性、重複性試驗；同時(shí)與基姐了于ASFV P30-2His6蛋白（N、C末端各融合1個H又雨is6标簽）的iELISA方法進(jìn)行獸醫臨姐哥床樣(yàng)本比較試驗。結果顯示：重組銀小ASFV P30蛋白和重組ASFV P30-2His北還6蛋白在Western-blot檢測吧的中，均能(néng)與豬ASFV陽性血清産生特異性雜交帶；基于重組藍說ASFV P30蛋白iELISA的最佳反應條件爲，抗原蛋白包被(bè鄉業i)質量濃度20 μg/mL、血清樣(yàng)品稀票友釋度1:1 000、酶标二抗稀釋度輛民1:40 000、血清樣(yàn市是g)品檢測OD450陽性結果臨界值0.長北22。該方法僅對(duì)AS遠花FV陽性血清呈特異性反應，1:3 200稀釋的陽性血清仍可檢出，批内試驗和會跳批間試驗變異系數均小于10%，可以消除His标簽所造成厭哥(chéng)的假陽性反應。本研究建立的ASFV P30 iELISA北通檢測方法爲ASFV抗體檢測提供了一種(zhǒng)有效手段。
Establishment and Ap相雨plication of the iELISA for De弟體tection of Antibodies against AS朋朋FV
In order to establish an indire爸說ct enzyme-linked 聽鄉immunosorbent assay（iELISA）for de懂文tecting antibodies against African sw工月ine fever virus（ASFV），the gene expres訊短sion engineering strain of recombinan資她t ASFV P30 was i是可nduced and expressed，follo兒校wed by purification and他筆 Western-blot detection of the re呢刀combinant proteins，then 美樂the iELISA method was established by t這船aking the purified recom小鄉binant proteins as antigens，its specif中北icity，sensitivity and repeatabil生用ity were subsequen她城tly evaluated. Meanwhile，it was co筆外mpared with the iELISA b商又ased on ASFV P30-2His6 prote笑雪in（one His6 tag wa購北s fused respectively做什 at N and C terminals）f媽問or veterinary clinical samp唱又les. The results showed that，by Weste門房rn-blot detection友司，both the recombinant proteins好女 of ASFV P30 and 雪做ASFV P30-2His6 暗民could produce specific hybrid 北一band with ASFV positive ser錢電um；the optimal reaction conditions文請 of iELISA based on 畫中the recombinant ASFV P30 prot市多eins were 20 μg/mL mass concentratio匠市n of antigen protein coating，1:1 00懂朋0 dilution of serum sample跳通s，1:40 000 dilution of goat anti-s地票wine IgG conjugate，and 0.22 crit制快ical value of positive serum samples OD小坐450. The established method was spec黑麗ific only to ASFV做秒 positive serum，1:3 也玩200 diluted positive ser電如um could still be dete制商cted，and the coefficients of v下明ariation（CV）of intra assay and 小業inter assay were新技 both less than 10%，which could elim話綠inate any false positive 著現reaction caused by His tag. Therefo文明re，an effective黑開 tool was provided fo海街r detecting antibodies against ASFV 藍畫by the established 知區ASFV P30 iELISA in the study.
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