【摘要】： 目的:應(yīng)用分子克隆技術(shù)獲得乙型肝炎病毒(HBV)B、C基因型X蛋白,為進(jìn)一步研究X基因在乙型肝炎病毒所致疾病中的作用奠定基礎(chǔ)。 方法: ①基因克隆:應(yīng)用PCR技術(shù),分別從本實驗室構(gòu)建的B、C基因型HBV保守的X基因真核表達(dá)重組子—pcDNA3.1-Xb和pcDNA3.1-Xc中獲取X全長基因,產(chǎn)物經(jīng)EcoR I和XhoI酶切,分別與原核表達(dá)載體pET-42a連接,轉(zhuǎn)化BL-21感受態(tài)細(xì)胞,抗生素篩選陽性重組子,PCR、酶切、測序鑒定。 ②蛋白表達(dá)純化:取鑒定后的B,C基因型X基因重組菌,IPTG誘導(dǎo)重組蛋白的表達(dá),SDS-PAGE電泳,用GST抗體經(jīng)Western Breeze化學(xué)發(fā)光法鑒定融合蛋白。優(yōu)化誘導(dǎo)時間和IPTG濃度,誘導(dǎo)重組蛋白大量表達(dá);低溫誘導(dǎo)可溶性X蛋白的形成。分別用GST和Ni-NTA bind純化試劑盒純化X蛋白,比較兩種方法的純化效果,并用Bradford法測定重組蛋白濃度。 結(jié)果: ①PCR法獲取的乙型肝炎病毒B、C基因型X基因的大小與預(yù)期一致;克隆后篩選的陽性重組子經(jīng)PCR、酶切、測序鑒定正確。 ②重組子均可被IPTG誘導(dǎo)表達(dá)融合蛋白,表達(dá)產(chǎn)物可被GST抗體識別;當(dāng)誘導(dǎo)時間為2 h、IPTG終濃度為0.5 mMol /L時,表達(dá)量均達(dá)最大,約為菌體總蛋白的40%以上;18℃,25℃,30℃均難以誘導(dǎo)可溶性蛋白的產(chǎn)生;溶解后的包涵體經(jīng)復(fù)性后進(jìn)行GST純化,但是最終未能得到目的蛋白;而用Ni-NTA純化后可直接得到大量融合蛋白,而且純度可達(dá)95%以上。 結(jié)論:成功構(gòu)建了B、C基因型乙型肝炎病毒X基因的原核表達(dá)系統(tǒng),并獲得高效表達(dá);重組蛋白經(jīng)Ni-柱純化后可得到大量重組蛋白,且純度較高,為進(jìn)一步研究X基因在乙型肝炎病毒所致疾病中的作用奠定基礎(chǔ)。
[Abstract]:Objective: to obtain the X protein of hepatitis B virus (HBV) B C genotype by molecular cloning technique, and to lay a foundation for further study on the role of X gene in hepatitis B virus induced diseases. Methods: 1 Gene cloning: using PCR technique, X full-length genes were obtained from pcDNA3.1-Xb and pcDNA3.1-Xc, the eukaryotic expression recombination of X gene conserved by HBV, which was constructed in our laboratory. The product was digested by EcoR I and XhoI, ligated with prokaryotic expression vector pET-42a, transformed into BL-21 receptive cells, screened for antibiotic positive recombinant, digested by PCR, and identified by sequencing. 2 protein expression and purification: the recombinant strain of X gene was obtained and the recombinant protein was induced by IPTG. SDS-PAGE electrophoresis was used to identify the fusion protein by Western Breeze chemiluminescence assay with GST antibody. The induction time and concentration of IPTG were optimized to induce the expression of recombinant protein and the formation of soluble X protein was induced by low temperature. X protein was purified by GST and Ni-NTA bind purification kit respectively. The purification effect of the two methods was compared and the concentration of recombinant protein was determined by Bradford method. Results: the size of genotype X gene of hepatitis B virus (HBV) obtained by 1PCR method was the same as expected, and the positive recombinant was digested by PCR, and sequenced. 2Recombinant could be induced by IPTG to express the fusion protein, and the expression product could be recognized by GST antibody, and when the induction time was 2 h, the final concentration of IPTG was 0.5 mMol / L, the expression reached the highest level, which was more than 40% of the total bacterial protein. It was difficult to induce the production of soluble protein at 18 鈩，

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