三元融合蛋白MBP-SERPINA3-IFN-κ的表达及抗单纯疱疹病毒/人乳头瘤病毒功能研究  

Prokaryotic expression and anti-HSV/HPV function of ternary fusion protein MBP-SERPINA3-IFN-κ

作　　者：冀旺盛 王斌 刘新奇[1] JI Wang-sheng;WANG Bin;LIU Xin-qi(State Key Laboratory of Medicinal Chemical Biology,School of Life Sciences,Nankai University,Tianjin 300071,China;不详)

机构地区：[1]药物化学生物学国家重点实验室南开大学生命科学学院,天津300071 [2]山西天南易联医疗科技有限公司,山西太原030000

出　　处：《中国病毒病杂志》2019年第6期443-448,共6页Chinese Journal of Viral Diseases

基　　金：国家自然科学基金(31870730)

摘　　要：目的原核表达三元融合蛋白MBP-SERPINA3-IFN-κ(简称MSIK),并在细胞和小鼠水平上对其抗人乳头瘤病毒(human papillomavirus,HPV)的功能进行研究。方法构建三元融合蛋白MSIK的原核表达质粒pET30-MBP-SERPINA3-IFN-κ,将构建好的质粒转入EL21(DE3)codon Plus大肠埃希菌并利用异丙基硫代半乳糖昔(isopropyl-β-D-thiogalactophyranoside,IPTG)诱导蛋白的表达,经亲和层析纯化得到蛋白,用聚丙烯酰胺凝胶电泳(SD&PAGE)验证目的蛋白的纯度,用1型单纯疱疹病毒(herpes simplex virus type 1,HSV-1)分别在角质细胞中及BALB/c小鼠上验证该蛋白的抗病毒活性,通过实时荧光定量PCR(Q-PCR)检测样本中病毒的含量。结果该融合蛋白经IPTG诱导表达并由亲和层析纯化后,获得相对分子质量约为100×10^3的纯化蛋白,经SDS-PAGE法检验,并使用Image J进行灰度分析,该融合蛋白纯度大于95%。在角质细胞中,该三元融合蛋白能够有效抑制HSV-1病毒的复制,在小鼠皮肤创伤模型中,该三元融合蛋白同样能够有效抑制HSV-1病毒的复制并能够促进创口的愈合。结论成功表达了三元融合蛋白MSIK,并利用亲和层析柱进行了有效的纯化。获得的融合蛋白具有良好的生物学活性,能够有效抑制HSV-1病毒的复制并促进创口的愈合。该三元融合蛋白可用于研发有效的抗HPV护创敷料,用来治疗一些由HPV引发的相关疾病。Objective To express the ternary fusion protein MBP-SERPINA3-IFN-κin prokaryotic cells and study its anti-HPV virus function at the cellular and mouse levels.Methods The recombinant plasmid pET30-MBP-SERPINA3-IFN-κcontaining gene encoding MBP-SERPINA3-IFN-κ(MSIK)was constructed and transformed into E.Coli.The recombinant E.Coli was cultured and induced by isopropyl-β-D-thiogalactoside(IPTG)and the fusion protein was purified using affinity chromatography column.SDS-PAGE method was used to verify the purity of the fusion protein.HSV-1 was used to test the antiviral activity of the fusion protein in human keratinocytes and BALB/c mice,respectively.The titer of virus in samples was determined by real-time fluorescent quantitative PCR(Q-PCR).Results The desired protein was purified by the affinity chromatography column and the relative molecular mass of the protein was approximately 100×10^3.SDSPAGE assay and Image J gray analysis showed that the purity of the fusion protein was more than 95%.The ternary fusion protein can effectively inhibit the replication of HSV-1 virus in keratinocytes.In mouse skin wound model,the ternary fusion protein was also effective in inhibiting HSV-1 virus replication and promoting wound healing.Conclusions The ternary fusion protein MSIK was successfully expressed and purified.The obtained fusion protein could inhibit the replication of HSV-1 virus and promote wound healing.The ternary fusion prothas the potential to develop anti-HPV drugs and prevent the diseases caused by HPV infection.

关 键 词：融合蛋白MBP-SERPINA3-IFN-κ 原核表达 亲和层析 人乳头瘤病毒 1型单纯疱疹病毒 促创口愈合 

分 类 号：R737[医药卫生—肿瘤]