融合基因重组BCG的构建及对肿瘤的抗性研究

发布时间：2018-03-04 08:37

本文选题：融合基因GCA　切入点：基因重组　出处：《青岛大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:将通过反转录PCR获得的EB病毒基因LMP2A与人源性GM-CSF基因进行融合,获得高效、稳定表达EB病毒的LMP2A抗原蛋白及GM-CSF因子的融合基因;将融合基因导入卡介苗(BCG),获得的重组BCG(r BCG)比对照常规BCG具有强的抗EB阳性肿瘤细胞的特性。方法:利用分子生物学技术中的RT-PCR方法分别获得EB病毒基因LMP2A与人GM-CSF基因编码序列的c DNA,用剪接式重叠延伸技术,将两段基因通过多肽接头(Gly4Ser)3DNA序列连接以获得融合基因GCA,将融合基因与已经获得的分泌型大肠杆菌-BCG穿梭表达载体p MV261连接,热激法转化E.coli.DH5α;利用质粒p MV261的Kan+筛选阳性菌株,并对阳性菌株分别用PCR扩增、双酶切及DNA测序鉴定;提纯重组质粒p MVGCA,将融合基因GCA与已经获得的分泌型BCG穿梭表达载体p MV261连接。利用电穿孔技术将重组质粒p MV261-GCA导入BCG,构建r BCG-GCA;利用质粒的抗性位点筛选阳性菌株,并对阳性菌株分别用PCR扩增和限制性酶切实验,检测目的基因的整合与转录,Middlebrook 7H9液体培养基大量培养制备r BCG-GCA,用Western Blot鉴定培养上清中GCA的融合蛋白表达。培养EB病毒阳性肿瘤细胞鼻咽癌细胞株SUNE1-LMP2细胞,制成肿瘤细胞单细胞悬液,用其建立BALB/C小鼠皮下EBV阳性肿瘤移植瘤模型,然后将r BCG-GCA菌株肿瘤局部皮下注射,于重组菌注射肿瘤细胞后第6、9、12、15、18、21天,观察并测量小鼠的肿瘤大小,设置PBS、p MV261载体、BCG组作为对照进行研究分析。成瘤时间以及肿瘤的体积、重量采用随机单因素方差分析(采用SPSS11.5软件)。两者之间进行t检验,以P0.05差异有统计学意义。结果:EB病毒基因LMP2A与人GM-CSF基因编码序列的c DNA经PCR鉴定与测序鉴定,与NCBI已报告的基因序列一致,将EB病毒基因LMP2A与人GM-CSF基因融合后,构建的融合基因经双酶切鉴定与测序鉴定,与理论值一致;将融合基因插入重组质粒p MV261,重组质粒p MV261经双酶切及测序鉴定插入正确,Ag85B基因和融合基因均能正确插入质粒p MV261,通过鉴定获得139bp的信号肽序列和1961bp的融合基因序列;将重组BCG培养后,取上清进行Western Blot分析,结果表明r BCG能分泌表达GM-CSF及LMP2A细胞因子;构建的重组BCG进行体内抗肿瘤实验,组成瘤时间最长(18d),与PBS组成瘤时间(8d)之间的差异有统计学意义(P0.05),r BCG对肿瘤的平均抑瘤率可达82%,与PBS组相比,差异有统计学意义(p0.05);结果表明重组BCG可抑制动物体内EB病毒阳性肿瘤细胞的生长。结论:EB病毒基因LMP2A与人源性GM-CSF融合基因GCG构建正确并能插入BCG分泌表达载体p MV261,电转化导入BCG后,重组质粒可在BCG中分泌性表达,表达的蛋白能被LMP2A抗体(或GM-CSF抗体)识别;重组BCG可抑制动物体内EB病毒阳性肿瘤细胞的生长;该实验为改造BCG、进行EB病毒相关肿瘤的免疫研究奠定了基础。
[Abstract]:Objective: to fuse EBV gene LMP2A obtained by reverse transcription PCR with human GM-CSF gene to obtain highly efficient and stable expression of EBV LMP2A antigen protein and GM-CSF factor fusion gene. When the fusion gene was introduced into BCG BCG, the recombinant BCG(r BCGG obtained was stronger than normal BCG in anti-EB positive tumor cells. Methods: Epstein-Barr virus (EBV) gene LMP2A and human GM-CSF were obtained by RT-PCR method in molecular biology. The c DNA of the gene encoding sequence, using splicing overlapping extension, The fusion gene was obtained by ligating the two segments of the gene through the peptide junction Gly4SerN 3 DNA sequence. The fusion gene was ligated with the secreted Escherichia coli -BCG shuttle expression vector p MV261, and transformed into E. coli.DH5 伪 by heat shock. The positive strains were screened by Kan of plasmid p MV261. The positive strains were amplified by PCR, digested by double enzyme and identified by DNA sequencing. The recombinant plasmid pMVGCA was purified, and the fusion gene GCA was ligated with the secreting BCG shuttle expression vector p MV261. The recombinant plasmid p MV261-GCA was introduced into BCGs by electroporation, and the rBCG-GCA was constructed, and the positive strains were screened by using the resistance sites of the plasmids. The positive strains were amplified by PCR and digested by restriction enzyme respectively. R BCG-GCA was prepared by mass culture of rBCG-GCA on the medium of integration and transcription of Middlebrook 7H9. The expression of GCA fusion protein in the supernatant was identified by Western Blot. SUNE1-LMP2 cells were cultured in nasopharyngeal carcinoma cell line, which was positive for Epstein-Barr virus (EBV). Single cell suspension of tumor cells was used to establish tumor transplantation model of subcutaneous EBV positive tumor in BALB/C mice. Then the tumor of r BCG-GCA strain was injected subcutaneously. The tumor size of mice was observed and measured on day 1821 after injection of tumor cells by recombinant bacteria. The tumorigenesis time, tumor volume and weight were analyzed by random univariate ANOVA (SPSS11.5 software). T test was performed between the two groups. Results the c DNA encoding sequence of LMP2A and GM-CSF gene were identified by PCR and sequenced, which were consistent with the sequence reported by NCBI. After fusion of LMP2A gene and GM-CSF gene, EB virus gene LMP2A was fused with human GM-CSF gene. The fusion gene was identified by double enzyme digestion and sequencing, which was consistent with the theoretical value. The fusion gene was inserted into the recombinant plasmid pMV261, and the recombinant plasmid p MV261 was confirmed by double enzyme digestion and sequencing. Both the correct Ag85B gene and the fusion gene could be inserted correctly into the plasmid pMV261. The signal peptide sequence of 139bp and the fusion gene sequence of 1961bp were obtained. After the recombinant BCG was cultured, the supernatant was extracted for Western Blot analysis. The results showed that r BCG secreted GM-CSF and LMP2A cytokines, and the recombinant BCG was used for anti-tumor experiment in vivo. The average tumor inhibitory rate of P0.05 BCG on tumor was 82% compared with that of PBS group, compared with that of PBS group, and the difference was significant between the longest tumor-forming time (18d) and PBS (8d), and the average inhibition rate of P0.05L BCG on tumor was 82d (P < 0.05), compared with that of PBS group. The results showed that the recombinant BCG could inhibit the growth of EBV positive tumor cells in animals. Conclusion the expression vector of BCG secreted by the fusion gene GCG of human GM-CSF and LMP2A of Epstein-EB virus can be constructed correctly and inserted into the expression vector of BCG secretion. MV261, after electroporation is imported into BCG, The recombinant plasmid can be secreted in BCG, the expressed protein can be recognized by LMP2A antibody (or GM-CSF antibody), and the recombinant BCG can inhibit the growth of EBV positive tumor cells in animal. This experiment laid a foundation for the immunological study of EB virus associated tumors.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.5

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