本文選題：MxA + PcDNA3.1��； 參考：《重慶醫(yī)科大學(xué)》2007年碩士論文

【摘要】： 第一部分人MxA基因重組真核載體的構(gòu)建及鑒定 目的:在前期獲得含有目的基因MxA的PAdtrack-MxA(含MxA的重組腺病毒穿梭質(zhì)粒)基礎(chǔ)上,應(yīng)用真核載體PcDNA3.1構(gòu)建重組真核載體PcDNA3.1-MxA。 方法:在大腸桿菌JM109中擴(kuò)增獲得含有目的基因MxA的PAdtrack-MxA和PcDNA3.1,使用兩者共同的限制性內(nèi)切酶Xba I、Not I進(jìn)行雙酶切,并連接以構(gòu)建重組真核載體PcDNA3.1-MxA,然后通過氨芐青霉素抗性篩選,雙酶切及PCR鑒定,選取鑒定正確的克隆測序,并應(yīng)用DNAssist 2.0軟件對序列與前期獲得的基因序列以及已知GenBank中MxA基因序列進(jìn)行同源性分析。 結(jié)果:經(jīng)限制性內(nèi)切酶、PCR鑒定重組真核載體PcDNA3.1-MxA構(gòu)建成功。測序結(jié)果表明本實驗所克隆片段長2012bp,包含人MxA基因序列,核苷酸序列分析表明與前期獲得的目的基因序列完全相同,而與GenBank中人MxA基因序列同源性也達(dá)99.75%,僅有5個堿基不同,且所編碼的氨基酸僅1個發(fā)生改變,此氨基酸位于MxA蛋白的非功能區(qū)。 結(jié)論:重組真核載體PcDNA3.1-MxA構(gòu)建成功,為下一步研究MxA抗乙型肝炎病毒作用奠定了基礎(chǔ)。 第二部分人MxA基因重組真核載體抗乙型肝炎病毒作用的實驗室研究 目的:體外研究PcDNA3.1-MxA體外抗HBV的作用,為進(jìn)一步研究體內(nèi)抗病毒及臨床應(yīng)用提供理論和實驗基礎(chǔ)。 方法:將構(gòu)建成功的重組真核載體PcDNA3.1-MxA轉(zhuǎn)染入HepG 2.2.15,根據(jù)PcDNA3.1-MxA新霉素抗性,使用G418抗性篩選出穩(wěn)定的細(xì)胞克隆。將HepG 2.2.15分為實驗組(穩(wěn)定表達(dá)PcDNA3.1-MxA的HepG 2.2.15)和對照組(未轉(zhuǎn)染組),應(yīng)用RT-PCR方法檢測兩組HepG2.2.15內(nèi)MxA mRNA水平的表達(dá),并應(yīng)用ELISA法檢測兩組HepG2.2.15內(nèi)HBsAg、HBeAg的水平。 結(jié)果:經(jīng)濃度梯度篩選顯示G418最適篩選濃度為600μg/ml,并在培養(yǎng)第3周篩選出穩(wěn)定表達(dá)PcDNA3.1-MxA的HepG 2.2.15。RT-PCR擴(kuò)增結(jié)果顯示實驗組的MxA mRNA水平均較對照組明顯升高,而且ELISA檢測結(jié)果顯示實驗組HBsAg、HBeAg水平明顯低于對照組,其結(jié)果具有顯著的統(tǒng)計學(xué)意義(P㩳0.01)。 結(jié)論:在HepG 2.2.15內(nèi)成功轉(zhuǎn)染并穩(wěn)定表達(dá)重組真核載體PcDNA3.1-MxA,PcDNA3.1-MxA能抑制HepG 2.2.15中HBV的復(fù)制及表達(dá)。
[Abstract]:Construction and Identification of Recombinant Eukaryotic Vector of Human MxA Gene Aim: to construct the recombinant eukaryotic vector PcDNA3.1-MxA by using the eukaryotic vector PcDNA3.1 on the basis of obtaining the recombinant adenovirus shuttle plasmid (Adtrack-MxA) containing the target gene MxA. Methods: PAdtrack-MxA and PcDNA3.1 containing the target gene MxA were amplified from Escherichia coli JM109. The recombinant eukaryotic vector PcDNA3.1-MxAwas ligated by using the restriction endonuclease Xba Ignit I for double digestion, and the recombinant eukaryotic vector PcDNA3.1-MxAwas then screened by ampicillin resistance. Double enzyme digestion and PCR identification were used to select the correct clone and sequencing, and DNAssist 2.0 software was used to analyze the homology of the sequence with the previously obtained gene sequence and the known MxA gene sequence in GenBank. Results: the recombinant eukaryotic vector PcDNA3.1-MxA was successfully constructed by restriction endonuclease polymerase chain reaction. The sequencing results showed that the cloned fragment was 2012bpand contained the human MxA gene sequence. The nucleotide sequence analysis showed that the sequence was identical to the target gene sequence obtained in the previous period, but the homology with the human MxA gene sequence in GenBank was 99.75, with only 5 bases different. Only one amino acid encoded changed, which was located in the non-functional region of MxA protein. Conclusion: the recombinant eukaryotic vector PcDNA3.1-MxA was successfully constructed, which laid a foundation for the further study of the anti-hepatitis B virus effect of MxA. The second part of the human MxA gene recombinant eukaryotic vector anti-hepatitis B virus in laboratory study Aim: to study the anti-HBV effect of PcDNA3.1-MxA in vitro and to provide theoretical and experimental basis for further study on anti-virus and clinical application in vivo. Methods: the recombinant eukaryotic vector PcDNA3.1-MxA was transfected into HepG 2.2.15. According to the PcDNA3.1-MxA neomycin resistance, the stable cell clones were screened by G418 resistance. HepG 2.2.15 was divided into experimental group (HepG 2.2.15 with stable expression of PcDNA3.1-MxA) and control group (untransfected group). The RT-PCR method was used to detect the expression of MxA mRNA in HepG2.2.15 and ELISA method was used to detect the level of HBAg-HBeAg in HepG2.2.15. Results: the optimal concentration of G418 was 600 渭 g / ml, and the HepG 2.2.15.RT-PCR amplification of stable expression of PcDNA3.1-MxA at the third week of culture showed that the MxA mRNA level of the experimental group was significantly higher than that of the control group. The results of ELISA test showed that the level of HBsAg HBeAg in the experimental group was significantly lower than that in the control group, and the result was significantly higher than that in the control group (P < 0.01). Conclusion: transfection and stable expression of recombinant eukaryotic vector PcDNA3.1-MxA1MxA in HepG 2.2.15 can inhibit the replication and expression of HBV in HepG 2.2.15.Conclusion: PcDNA3.1-MxA can inhibit the replication and expression of PcDNA3.1-MxA in HepG 2.2.15.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R346

【相似文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 沈欽海;木芙蓉在肝病中的藥理作用及機(jī)理研究[D];重慶醫(yī)科大學(xué);2006年

相關(guān)碩士學(xué)位論文 前1條

1 黃琴;人MxA基因重組真核載體抗乙型肝炎病毒作用的實驗室研究[D];重慶醫(yī)科大學(xué);2007年

，

本文編號：1920255