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目的:构建重组厌氧芽胞梭菌saccharobutylicum内源性β-1,4葡聚糖酶启动子(eglA p人)-表皮生长因子受体2胞外区(hHer2/neu ECD人)-白细胞介素12(rhIL-12)融合基因穿梭表达载体(pIMP1 eglA p-hHer2/neu ECD-rhIL-12),为进一步制备eglAp-hHer2/neu-rhIL-12融合基因修饰的厌氧芽胞梭菌,并探讨其对Her2/neu+实体肿瘤的基因治疗作用奠定基础。方法:以含有全长hHer2/neu序列的真核表达质粒(pcDNA3.1 hHer2/neu)为模板,采用PCR方法,扩增hHer2/neu ECD基因片段;将其插入重组rhIL-12真核表达质粒pcD-NA6 rhIL-12(p1)中的rhIL-12基因上游,获得pcDNA6 hHer2/neu ECD-rhIL-12(p2)真核表达质粒;设计合成eglAp基因中一段55 bp序列作为模板,通过连续PCR的方法,扩增eglAp片段(335 bp),并构建T-eglA p(p3)亚克隆质粒;通过酶切连接的方法,将eglA p片段插入p2质粒中的hHer2/neu ECD基因上游,构建pcDNA6 eglA p-hHer2/neu ECD-rhIL-12(p4)真核表达质粒;通过in-fusion技术,将融合基因eglAp-hHer2/neu ECD-rhIL-12插入pIMP1穿梭表达质粒,构建重组pIMP1eglA p-hHer2/neu ECD-rhIL-12(p5)穿梭表达载体,并进行菌液PCR、酶切及测序鉴定。结果:获得的hHer2/neu ECD和eglA p片段及p2、p3、p4重组质粒、p5融合基因重组穿梭表达载体,其PCR产物和酶切片段大小与预期一致,测序结果证实各基因序列与GenBank中mRNA或DNA序列一致。结论:成功地构建了大肠杆菌-厌氧芽胞梭菌融合基因穿梭表达载体(pIMP1 eglAp-hHer2/neu ECD-rhIL-12),为进一步制备eglAp-hHer2/neu ECD-rhIL-12融合基因修饰的厌氧芽胞梭菌,探讨其对Her2/neu+实体肿瘤的基因治疗作用奠定基础。
Objective: To construct the recombinant endogenous β-1,4 glucanase promoter of saccharobutylicum (eglA p human) - epidermal growth factor receptor 2 extracellular domain (hHer2 / neu ECD) - interleukin 12 (rhIL-12) fusion gene shuttle expression vector (pIMP1 eglA p-hHer2 / neu ECD-rhIL-12), in order to further prepare eglAp-hHer2 / neu-rhIL-12 fusion gene modified Clostridium perfringens It laid the foundation for gene therapy of Her2 / neu + solid tumors. METHODS: The hHer2 / neu ECD gene fragment was amplified by PCR from the eukaryotic expression plasmid containing the full-length hHer2 / neu sequence (pcDNA3.1 hHer2 / neu) and inserted into the recombinant rhIL-12 eukaryotic expression plasmid The pcDNA6 hHer2 / neu ECD-rhIL-12 (p2) eukaryotic expression plasmid was obtained upstream of the rhIL-12 gene in pcD-NA6 rhIL-12 (p1). A 55 bp sequence of eglAp gene was designed and synthesized as a template. , The eglAp fragment (335 bp) was amplified and the T-eglA p (p3) subclone plasmid was constructed. The eglA p fragment was inserted upstream of the hHer2 / neu ECD gene in the p2 plasmid by restriction enzyme ligation to construct pcDNA6 eglA p-hHer2 / neu ECD-rhIL-12 (p4) eukaryotic expression plasmid. The fusion gene eglAp-hHer2 / neu ECD-rhIL-12 was inserted into pIMP1 shuttle expression plasmid by in-fusion technology to construct recombinant pIMP1eglA p-hHer2 / neu ECD-rhIL-12 (p5) shuttle expression vector, and bacterial liquid PCR, digestion and sequencing identification. Results: The recombined hHer2 / neu ECD and eglA p fragments, p2, p3 and p4 recombinant plasmids and p5 fusion gene shuttle vector were constructed. The PCR products and the size of the fragments were as expected. The sequencing results confirmed that the sequences of these genes were identical with those of GenBank mRNA or DNA sequence. Conclusion: The E. coli-Clostridium aeruginosa fusion gene shuttle expression vector (pIMP1 eglAp-hHer2 / neu ECD-rhIL-12) was constructed successfully. In order to further improve the expression of eglAp-hHer2 / neu ECD-rhIL-12 fusion gene Clostridium perfringens, to explore its role in Her2 / neu + solid tumor gene therapy foundation.