本文選題：ACBD + 真核表達(dá)載體�。� 參考：《中國病原生物學(xué)雜志》2017年08期

【摘要】：目的構(gòu)建ACBD3基因片段真核表達(dá)載體并表達(dá)蛋白,為研究其與肺炎衣原體包涵體膜蛋白Cpn0308/Cpn0147相互作用的分子機(jī)制奠定基礎(chǔ)。方法根據(jù)與肺炎衣原體包涵體膜蛋白Cpn0308/Cpn0147作用的配體核苷酸序列設(shè)計(jì)引物,以HeLa細(xì)胞cDNA為模板,通過PCR獲得ACBD3基因片段,與pcDNA3.1+/Flag質(zhì)粒經(jīng)雙酶切后在T4連接酶作用下連接,構(gòu)建表達(dá)載體pcDNA3.1+/Flag-ACBD3,經(jīng)菌落PCR、雙酶切及質(zhì)粒測序鑒定后轉(zhuǎn)染HeLa細(xì)胞,采用Western blot與間接免疫熒光法檢測目的蛋白表達(dá)。結(jié)果 PCR擴(kuò)增目的基因片段約883bp,與預(yù)期相符。經(jīng)菌落PCR、雙酶切驗(yàn)證及測序分析重組質(zhì)粒構(gòu)建成功。間接免疫熒光法檢測重組質(zhì)粒轉(zhuǎn)染后的HeLa細(xì)胞,觀察到表達(dá)的蛋白位于細(xì)胞胞漿;SDS-PAGE檢測表達(dá)蛋白分子質(zhì)量單位(Mr)為33.5×10~3,與預(yù)期相符。結(jié)論成功構(gòu)建了pcDNA3.1+/Flag-ACBD3真核表達(dá)載體并實(shí)現(xiàn)目的蛋白的表達(dá),為進(jìn)一步研究其生物學(xué)功能及其與肺炎衣原體包涵體膜蛋白Cpn0308/Cpn0147之間的相互作用奠定了基礎(chǔ)。
[Abstract]:Objective to construct the eukaryotic expression vector of ACBD3 gene fragment and express the protein in order to study the molecular mechanism of its interaction with chlamydia pneumoniae inclusion body membrane protein (Cpn0308/Cpn0147). Methods according to the ligand nucleotide sequence of chlamydia pneumoniae inclusion body membrane protein (Cpn0308/Cpn0147), the ACBD3 gene fragment was obtained by PCR using HeLa cell cDNA as template. The ACBD3 gene fragment was digested with pcDNA3.1 / Flag plasmid and ligated with T4 ligand. The expression vector pcDNA3.1 / Flag-ACBD3 was constructed and identified by colony PCR, double enzyme digestion and plasmid sequencing. The expression of the target protein was detected by Western blot and indirect immunofluorescence assay. Results the target gene fragment amplified by PCR was 883 BP, which was consistent with the expectation. The recombinant plasmid was successfully constructed by colony PCR, double enzyme digestion and sequencing analysis. Indirect immunofluorescence assay was used to detect the HeLa cells transfected with recombinant plasmids. It was observed that the expressed protein was located in the cytoplasmic SDS-PAGE and the molecular weight unit of the expressed protein was 33.5 脳 10 ~ (-3), which was consistent with the expectation. Conclusion the eukaryotic expression vector of pcDNA3.1 / Flag-ACBD3 was successfully constructed and the expression of target protein was realized, which laid a foundation for further study of its biological function and its interaction with chlamydia pneumoniae inclusion body membrane protein Cpn0308/Cpn0147.
【作者單位】： 河北北方學(xué)院病原生物學(xué)與免疫研究所臨床檢驗(yàn)診斷學(xué)重點(diǎn)實(shí)驗(yàn)室;
【基金】：河北省自然科學(xué)基金項(xiàng)目(No.C2014405041)
【分類號(hào)】：R374

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本文編號(hào)：1968936