本文選題：金黃色葡萄球菌　切入點：CTB-IsdBid-Clfais蛋白　出處：《中國畜牧獸醫(yī)》2017年12期 　論文類型：期刊論文

【摘要】：為了研究含有分子內(nèi)佐劑的CTB-IsdBid-Clfais(CIC)蛋白表達及其免疫活性,試驗采用重疊PCR方法將分子內(nèi)佐劑CTB與IsdBid-Clfais基因串聯(lián),并將CTB-IsdBid-Clfais(CIC)插入到表達載體pET-32a(+)中,構(gòu)建pET-32a(+)-CTB-IsdBid-Clfais重組質(zhì)粒,將鑒定正確的重組質(zhì)粒轉(zhuǎn)化到大腸桿菌BL21感受態(tài)細胞中表達目的蛋白CIC,利用Western blotting方法對其進行鑒定,并以ELISA方法檢測CIC蛋白的免疫活性。結(jié)果發(fā)現(xiàn),試驗成功擴增了2 072bp的目的基因CIC,并將CIC正確連接到pET-32a(+)載體上,構(gòu)建了pET-32a(+)-CTB-IsdBid-Clfais重組質(zhì)粒;Western blotting結(jié)果證實,該重組質(zhì)粒能夠在大腸桿菌BL21感受態(tài)細胞中正確表達CIC蛋白,分子質(zhì)量大小為95.9ku;ELISA結(jié)果顯示,CIC試驗組與IsdBid、Clfais蛋白組間均無顯著差異(P0.05),與BSA組間差異極顯著(P0.01)。綜上所述,本研究成功構(gòu)建了pET-32a(+)-CTB-IsdBid-Clfais重組質(zhì)粒,該質(zhì)粒在大腸桿菌BL21中成功表達了CIC蛋白,且CIC能與IsdBid、Clfais免疫小鼠血清發(fā)生反應,具有較強的免疫活性。
[Abstract]:In order to study the expression and immunological activity of CTB-IsdBid-Clfais-CICs containing intramolecular adjuvants, the recombinant plasmid pET-32a (-CTB-IsdBid-Clfais) was constructed by using overlapping PCR method to connect the intramolecular adjuvant CTB with the IsdBid-Clfais gene and insert CTB-IsdBid-Clfais into the expression vector pET-32a (pET-32a), and construct the recombinant plasmid pET-32a (-CTB-IsdBid-Clfais), and construct the recombinant plasmid pET-32a (-CTB-IsdBid-Clfais). The identified recombinant plasmid was transformed into Escherichia coli BL21 competent cells to express the target protein CIC. Western blotting method was used to identify the recombinant plasmid, and the ELISA method was used to detect the immunological activity of CIC protein. The target gene CIC-2072 BP was amplified successfully, and the CIC was correctly ligated to pET-32a () vector. The recombinant plasmid pET-32a (-CTB-IsdBid-Clfais) was constructed by Western blotting. The results showed that the recombinant plasmid could correctly express CIC protein in Escherichia coli BL21 competent cells. The results of Elisa showed that there was no significant difference between CIC test group and Isd Bidn Clfais protein group, but there was very significant difference between CICC test group and BSA group. In conclusion, the recombinant plasmid pET-32a was successfully constructed in this study. The plasmid successfully expressed CIC protein in Escherichia coli BL21, and CIC could react with the serum of Isd Bidsil-Clfais immunized mice, and had strong immune activity.
【作者單位】： 黑龍江八一農(nóng)墾大學生命科學技術(shù)學院;
【基金】：黑龍江省大學生創(chuàng)新創(chuàng)業(yè)訓練計劃項目(201510223001) 黑龍江省自然科學基金項目(C201443) 大慶市指導性科技計劃項目(S2dfy-2015-44)
【分類號】：S852.611

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本文編號：1612751