携带NK4与IL2基因真核表达载体的减毒沙门氏菌的构建.DOCVIP

临床医学论文-携带NK4与IL2基因真核表达载体的减毒沙门氏菌的构建 【摘要】? 目的： 构建携带NK4基因和IL2基因真核表达载体的减毒沙门氏菌株. 方法： 用基因工程方法将NK4基因和IL2基因克隆入真核表达载体pcDNA4，并进行基因测序. 重组质粒经鉴定后再电转入减毒沙门氏菌Ty21a中，通过PCR和酶切鉴定，并对构建的重组减毒沙门氏菌进行体外稳定性观察. 结果： 经PCR和酶切证实，构建了分别含NK4基因和IL2基因的重组真核表达质粒pcDNA4NK4和pcDNA4IL2，并将他们成功导入减毒沙门氏菌Ty21a中. 稳定性实验结果表明该重组菌株在体外能稳定地繁殖、生长和传代. 结论: 成功构建携带NK4基因和IL2基因真核表达载体的减毒沙门氏菌株，为探索制备携带NK4和IL2基因的肿瘤减毒活疫苗奠定了基础. 【关键词】? NK4;基因；白细胞介素2；沙门菌属；疫苗，减毒 【Abstract】 AIM: To construct two recombinant attenuated Salmonella typhimurium strains carrying NK4 and IL2 gene eukaryotic expression vector. METHODS: NK4 gene and IL2 gene were obtained by PCR, then cloned into eukaryotic expression vector pcDNA4. The recombinants were identified, and transformed into attenuated Salmonella typhimurium strain Ty21a, and the clones were screened by PCR and restriction enzyme digestion. The stability of the two recombinant attenuated salmonella typhimurium strains was evaluated. RESULTS: Confirmed by PCR and restriction enzyme digestion, recombinant eukaryotic expression plasmid pcDNA4NK4 and plasmid pcDNA4IL2 were constructed successfully, and then introduced into an attenuated Salmonella typhimurium Ty21a. The recombinant strains stably reproduced, grew and passaged in vitro. CONCLUSION: The recombinant attenuated Salmonella typhimurium strains carrying plasmid pcDNA4NK4 and plasmid pcDNA4IL2 can be successfully constructed and identified, and it will help to develop a tumor expressing recombinant live vaccine carrying NK4 and IL2. 　　【Keywords】 NK4; genes; interleukin2; Salmonella; vaccines, attenuated 　　0? 引言??? 　　肝细胞生长因子(hepatocyte growth factor, HGF)是由间质细胞产生的多功能细胞因子，具有强促分裂、组织形成、诱导上皮细胞迁移，侵袭及诱导血管生成等作用,这些都有利于肿瘤组织的发生与发展［1］. 1997年Date等［2］用胰弹性蛋白酶对重组人HGF进行消化裂解，发现了一种新的全面拮抗HGF生物活性功能的拮抗剂NK4. NK4作为HGF的拮抗剂能显著抑制肿瘤细胞的生长、增殖和转移, 在肿瘤基因治疗中有潜在的应用前景.??? 　　白细胞介素2 (interleukin2, IL2)又称T细胞生长因子，是机体免疫调节中的重要细胞因子之一，可通过提高自然杀伤细胞与LAK细胞活性，增强细胞毒性T细胞(cytotoxic tlymphocyte, CTL)对肿瘤细胞的杀伤作用，并激活肿瘤侵润淋巴细胞从而诱导机体的抗肿瘤免疫反应，使肿瘤生长停止或瘤体减小［3］. 随着分子生物学的发展，IL2基因