本文選題：融合基因GCA　切入點：基因重組　出處：《青島大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:將通過反轉(zhuǎn)錄PCR獲得的EB病毒基因LMP2A與人源性GM-CSF基因進行融合,獲得高效、穩(wěn)定表達EB病毒的LMP2A抗原蛋白及GM-CSF因子的融合基因;將融合基因?qū)肟ń槊?BCG),獲得的重組BCG(r BCG)比對照常規(guī)BCG具有強的抗EB陽性腫瘤細(xì)胞的特性。方法:利用分子生物學(xué)技術(shù)中的RT-PCR方法分別獲得EB病毒基因LMP2A與人GM-CSF基因編碼序列的c DNA,用剪接式重疊延伸技術(shù),將兩段基因通過多肽接頭(Gly4Ser)3DNA序列連接以獲得融合基因GCA,將融合基因與已經(jīng)獲得的分泌型大腸桿菌-BCG穿梭表達載體p MV261連接,熱激法轉(zhuǎn)化E.coli.DH5α;利用質(zhì)粒p MV261的Kan+篩選陽性菌株,并對陽性菌株分別用PCR擴增、雙酶切及DNA測序鑒定;提純重組質(zhì)粒p MVGCA,將融合基因GCA與已經(jīng)獲得的分泌型BCG穿梭表達載體p MV261連接。利用電穿孔技術(shù)將重組質(zhì)粒p MV261-GCA導(dǎo)入BCG,構(gòu)建r BCG-GCA;利用質(zhì)粒的抗性位點篩選陽性菌株,并對陽性菌株分別用PCR擴增和限制性酶切實驗,檢測目的基因的整合與轉(zhuǎn)錄,Middlebrook 7H9液體培養(yǎng)基大量培養(yǎng)制備r BCG-GCA,用Western Blot鑒定培養(yǎng)上清中GCA的融合蛋白表達。培養(yǎng)EB病毒陽性腫瘤細(xì)胞鼻咽癌細(xì)胞株SUNE1-LMP2細(xì)胞,制成腫瘤細(xì)胞單細(xì)胞懸液,用其建立BALB/C小鼠皮下EBV陽性腫瘤移植瘤模型,然后將r BCG-GCA菌株腫瘤局部皮下注射,于重組菌注射腫瘤細(xì)胞后第6、9、12、15、18、21天,觀察并測量小鼠的腫瘤大小,設(shè)置PBS、p MV261載體、BCG組作為對照進行研究分析。成瘤時間以及腫瘤的體積、重量采用隨機單因素方差分析(采用SPSS11.5軟件)。兩者之間進行t檢驗,以P0.05差異有統(tǒng)計學(xué)意義。結(jié)果:EB病毒基因LMP2A與人GM-CSF基因編碼序列的c DNA經(jīng)PCR鑒定與測序鑒定,與NCBI已報告的基因序列一致,將EB病毒基因LMP2A與人GM-CSF基因融合后,構(gòu)建的融合基因經(jīng)雙酶切鑒定與測序鑒定,與理論值一致;將融合基因插入重組質(zhì)粒p MV261,重組質(zhì)粒p MV261經(jīng)雙酶切及測序鑒定插入正確,Ag85B基因和融合基因均能正確插入質(zhì)粒p MV261,通過鑒定獲得139bp的信號肽序列和1961bp的融合基因序列;將重組BCG培養(yǎng)后,取上清進行Western Blot分析,結(jié)果表明r BCG能分泌表達GM-CSF及LMP2A細(xì)胞因子;構(gòu)建的重組BCG進行體內(nèi)抗腫瘤實驗,組成瘤時間最長(18d),與PBS組成瘤時間(8d)之間的差異有統(tǒng)計學(xué)意義(P0.05),r BCG對腫瘤的平均抑瘤率可達82%,與PBS組相比,差異有統(tǒng)計學(xué)意義(p0.05);結(jié)果表明重組BCG可抑制動物體內(nèi)EB病毒陽性腫瘤細(xì)胞的生長。結(jié)論:EB病毒基因LMP2A與人源性GM-CSF融合基因GCG構(gòu)建正確并能插入BCG分泌表達載體p MV261,電轉(zhuǎn)化導(dǎo)入BCG后,重組質(zhì)粒可在BCG中分泌性表達,表達的蛋白能被LMP2A抗體(或GM-CSF抗體)識別;重組BCG可抑制動物體內(nèi)EB病毒陽性腫瘤細(xì)胞的生長;該實驗為改造BCG、進行EB病毒相關(guān)腫瘤的免疫研究奠定了基礎(chǔ)。
[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 偽 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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R730.5

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