本文選題：LIM礦化蛋白　切入點(diǎn)：腺病毒　出處：《重慶醫(yī)科大學(xué)》2007年碩士論文

【摘要】： 目的:應(yīng)用AdEasy腺病毒載體系統(tǒng),構(gòu)建人LMP-1基因的腺病毒重組體,感染體外培養(yǎng)的兔BMSCs,鑒定細(xì)胞對(duì)LMP-1的表達(dá)情況,為進(jìn)一步動(dòng)物實(shí)驗(yàn)奠定基礎(chǔ)。 方法:采用PCR的方法以pIRES2-EGFP-LMP-1質(zhì)粒為模板進(jìn)行擴(kuò)增,在下游引物中加入特定序列,使擴(kuò)增出的LMP-1基因帶有編碼6個(gè)組氨酸(即His標(biāo)簽)的堿基序列。將得到的帶有His標(biāo)簽堿基序列的LMP-1基因作為目的基因,通過(guò)TA克隆與pMD18-T載體連接并測(cè)序。雙酶切后插入至腺病毒穿梭質(zhì)粒pAdtrack-CMV內(nèi)。PmeⅠ線性化陽(yáng)性克隆pAdtrack-LMP-1,在BJ5183菌內(nèi)完成與骨架質(zhì)粒pAdeasy-1的同源重組,構(gòu)建出重組腺病毒質(zhì)粒pAd-LMP-1。通過(guò)脂質(zhì)體介導(dǎo)在HEK293細(xì)胞內(nèi)包裝出復(fù)制缺陷的重組腺病毒Ad-LMP-1,大量擴(kuò)增、純化并測(cè)定滴度。 結(jié)合骨髓密度梯度離心法和貼壁篩選法從兔骨髓中分離培養(yǎng)BMSCs。重組腺病毒以不同的MOI感染第三代BMSCs,在顯微鏡下觀察其生長(zhǎng)情況及感染效率。然后以最佳MOI感染BMSCs,3天后收集細(xì)胞行RT-PCR法檢測(cè)LMP-1基因的mRNA表達(dá),再利用抗His標(biāo)簽的特異抗體以Western Blot的方法鑒定LMP-1基因的蛋白表達(dá)。 結(jié)果:測(cè)序鑒定攜帶His標(biāo)簽的LMP-1基因的重組質(zhì)粒pMD18-T構(gòu)建成功。成熟的體外分離培養(yǎng)BMSCs的方法方便簡(jiǎn)單,收獲細(xì)胞數(shù)量多且增殖能力強(qiáng)。重組腺病毒以150的MOI值感染兔BMSCs可以獲得最佳的感染效率,即在3天后達(dá)到50%～70%。收集病毒感染后的BMSCs行RT-PCR檢測(cè)表明存在LMP-1基因的mRNA表達(dá),Western Blot亦證實(shí)LMP-1基因的蛋白表達(dá)。 結(jié)論:成功構(gòu)建攜帶His標(biāo)簽的人LIM礦化蛋白-1(LMP-1)基因的腺病毒重組體。證實(shí)重組病毒感染BMSCs后細(xì)胞能有效地表達(dá)LMP-1,為L(zhǎng)MP-1動(dòng)物體內(nèi)的實(shí)驗(yàn)研究提供了依據(jù)。
[Abstract]:Objective: to construct adenovirus recombinant of human LMP-1 gene by AdEasy adenovirus vector system, and infect rabbit BMSCs in vitro, identify the expression of LMP-1, and lay a foundation for further animal experiments.
Methods: the PCR method using pIRES2-EGFP-LMP-1 plasmid as template for amplification, adding specific sequence in the downstream primers, the LMP-1 gene was amplified with encoding 6 histidine (His tag) of the base sequence. The His tagged nucleotide sequence of the LMP-1 gene as the target gene, was cloned by TA and pMD18-T vector. Double enzyme digestion and sequencing. After inserted into Adenovirus Shuttle Plasmid pAdtrack-CMV.Pme 1 linear positive clone pAdtrack-LMP-1 BJ5183 in bacteria within homologous recombination with plasmid pAdeasy-1, the recombinant adenovirus plasmid pAd-LMP-1. by liposome mediated package of recombinant adenovirus Ad-LMP-1 replication defective in HEK293 cells proliferation purification, and the titer was determined.
And adherence screening method from rabbit bone marrow in the separation of the third generation BMSCs were infected with different MOI BMSCs. recombinant adenovirus combined with cultured bone marrow by density gradient centrifugation, to observe the growth and infection efficiency under the microscope. Then the optimal MOI BMSCs infection, collected 3 days after the expression of RT-PCR was detected by LMP-1 gene of mRNA cell line, and then the use of specific antibody anti His tag with LMP-1 Western Blot method for identification of gene expression.
Results: the recombinant plasmid pMD18-T LMP-1 gene sequencing with His tag were successfully constructed. The mature in vitro culture method of BMSCs is convenient and simple, the cells were harvested and a large number of strong proliferation ability. The recombinant adenovirus with MOI values of 150 rabbits infected with BMSCs can get the best effect of infection rate reached 50% ~ 70%. BMSCs collection virus RT-PCR detection showed that the expression of LMP-1 gene mRNA in 3 days, Western Blot also confirmed the expression of LMP-1 protein.
Conclusion: the adenovirus recombinant carrying human His LIM protein -1 (LMP-1) gene was successfully constructed. It is proved that the recombinant LMP-1 can effectively reach LMP-1 after infection with BMSCs, which provides a basis for the experimental research of LMP-1 animal.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 江遜,崔鵬程,陳文弦,趙大慶,張殿忠,張志培;人骨髓間充質(zhì)干細(xì)胞體外培養(yǎng)及生物特性的觀察[J];第四軍醫(yī)大學(xué)學(xué)報(bào);2003年04期

2 劉素彩,趙志剛,趙長(zhǎng)安,張志勇,劉靖,李恩;LMP-1 mRNA反義寡核苷酸抑制大鼠成骨細(xì)胞的分化[J];基礎(chǔ)醫(yī)學(xué)與臨床;2002年02期

3 孫佳,鹿培源,賈弘y，

本文編號(hào)：1694003