【摘要】：根據(jù)豬肺炎支原體168(Mycoplasma hyopneumoniae 168,Mhp168)弱毒株p46和p65基因序列設(shè)計(jì)特異性引物,進(jìn)行PCR擴(kuò)增并構(gòu)建表達(dá)載體pET-28a-p46,pET-28a-p65以及pET-28a-p46-p65。以表達(dá)載體pET-28a-p46和pET-28a-p46-p65為模板,將其中p46基因3個TGA密碼子突變?yōu)門GG。原核表達(dá)并純化的蛋白與佐劑以體積比1∶1乳化后免疫小鼠,通過酶聯(lián)免疫吸附反應(yīng)(ELISA)測定小鼠血清的抗體效價。分別選取抗體效價較高的小鼠進(jìn)行攻毒試驗(yàn)。經(jīng)間接ELISA試驗(yàn)證明單蛋白P46、P65以及融合蛋白P46-P65的血清效價分別為:1∶1 280000,1∶128 000和1∶2 560 000;攻毒試驗(yàn)證明,P46、P65單蛋白和P46-P65融合蛋白疫苗的保護(hù)率分別可達(dá)82%,78%和98%。融合蛋白P46-P65具有良好的免疫原性,并且要優(yōu)于單蛋白P46和P65的免疫原性。
[Abstract]:According to the sequence of p46 and p65 genes of attenuated strain p46 and p65 of Mycoplasma pneumoniae 168 (Mycoplasma hyopneumoniae 168 Mhp168), specific primers were designed for PCR amplification and construction of expression vector pET-28a-p46,pET-28a-p65 and pET-28a-p46-p65.. Using the expression vectors pET-28a-p46 and pET-28a-p46-p65 as templates, three TGA codon of p46 gene were mutated into TGG.. Mice were immunized with prokaryotic protein and adjuvant after emulsified at 1:1. The antibody titers of mouse serum were determined by Elisa (ELISA). Mice with high antibody titer were selected to test the antivirus. The results of indirect ELISA test showed that the serum titers of single protein P46, P65 and fusion protein P46-P65 were 1:1 2800001: 128000 and 1:2 560000, respectively. The protective rates of P46 P65 and P46-P65 fusion protein vaccine were up to 82% and 98%, respectively. The fusion protein P46-P65 has good immunogenicity and is superior to that of P46 and p65.
【作者單位】： 河北師范大學(xué)生命科學(xué)學(xué)院;
【基金】：河北省科技支撐計(jì)劃資助項(xiàng)目(13226603D)
【分類號】：S852.62

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本文編號：2370937