本文選題：HLX + 原核表達(dá)�。� 參考：《江蘇大學(xué)》2009年碩士論文

【摘要】： 目的: 克隆人HLX(H2.0-like homeobox gene,HLX)基因,原核表達(dá)并純化HLX蛋白;制備針對人HLX融合蛋白的兔源性多克隆抗體并加以鑒定;定量PCR檢測胃癌病人外周血單個(gè)核細(xì)胞中HLX mRNA的表達(dá)水平,以探討HLX基因表達(dá)水平對機(jī)體Th1/Th2平衡狀態(tài)的影響及其與胃癌發(fā)生發(fā)展的關(guān)系。 方法: (1)采用RT-PCR方法從人臍血單個(gè)核細(xì)胞獲得HLX基因,克隆至pMD19-T載體,轉(zhuǎn)化E.coli DH5α宿主菌,挑取菌落進(jìn)行酶切鑒定,將初步確定的陽性克隆進(jìn)一步測序確認(rèn)。 (2)以PCR的方法從HLX陽性克隆中獲得HLX基因的ORF區(qū)全長,連接至pQE30原核表達(dá)載體,轉(zhuǎn)化E.coli DH5α宿主菌,挑取菌落進(jìn)行酶切和測序鑒定。 (3)將陽性重組質(zhì)粒pQE30-HLX轉(zhuǎn)化入E.coli M15菌中,經(jīng)異丙基-β-D-硫代半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)誘導(dǎo)表達(dá),所表達(dá)的HLX融合蛋白經(jīng)SDS-PAGE電泳及Western blot鑒定。大量誘導(dǎo)表達(dá)HLX融合蛋白,利用Profinity IMAC Ni-Charged Resin親和層析柱分離純化,以HLX蛋白作為抗原免疫家兔,收集含抗HLX多克隆抗體的兔血清,并用ELISA和Westernblot法鑒定抗體的效價(jià)和特異性。 (4)定量PCR檢測胃癌患者外周血單個(gè)核細(xì)胞(peripheral blood mononuclearcells,PBMC)中HLX的表達(dá),并與健康對照組進(jìn)行比較研究,同時(shí)分析胃癌患者HLX的表達(dá)與T-bet、GATA-3表達(dá)的關(guān)系,以從轉(zhuǎn)錄因子水平了解可能存在于胃癌患者機(jī)體的Th1/Th2平衡失調(diào)狀態(tài)。 結(jié)果: (1)成功克隆了人HLX基因,構(gòu)建了pMD19-T-HLX質(zhì)粒。 (2)成功克隆人HLX基因ORF區(qū)全長,構(gòu)建pQE30-HLX原核表達(dá)質(zhì)粒。測序結(jié)果顯示,連接至pQE30載體的目的片段為HLX的ORF區(qū),全長1467bp,編碼488個(gè)氨基酸殘基,與GenBank中發(fā)表的序列完全一致。經(jīng)誘導(dǎo)和純化獲得HLX融合蛋白,大小約為55kD。 (3)成功制備了針對人HLX蛋白的多克隆抗體,經(jīng)ELISA和Western blot鑒定,具有良好的反應(yīng)性及特異性,也表明HLX融合蛋白具有良好的免疫原性。 (4)實(shí)時(shí)熒光定量PCR檢測了胃癌患者PBMC的HLX表達(dá)水平,較之健康對照組,其表達(dá)量有所下降。同法檢測了胃癌患者T-bet、GATA-3表達(dá)的情況,分析了與兩種基因間的相關(guān)性。 結(jié)論: 成功構(gòu)建了pQE30-HLX原核表達(dá)質(zhì)粒并在大腸埃希菌中有效表達(dá);獲得人HLX融合蛋白,并以此為抗原,制備了特異性多克隆抗體;檢測了HLX在胃癌患者PBMC的表達(dá)水平,較之健康對照組有所下降,并其表達(dá)水平與其他Th1型細(xì)胞因子或轉(zhuǎn)錄因子T-bet呈正相關(guān),與GATA-3呈負(fù)相關(guān),推測其可能與胃癌的發(fā)生發(fā)展存在一定的關(guān)系。
[Abstract]:Objective: Cloning of human HLX(H2.0-like homeobox gene, prokaryotic expression and purification of HLX protein, preparation and identification of rabbit polyclonal antibody against human HLX fusion protein, quantitative PCR detection of HLX mRNA expression in peripheral blood mononuclear cells of gastric cancer patients, To investigate the effect of HLX gene expression level on the balance of Th1/Th2 and its relationship with the occurrence and development of gastric cancer. Methods: 1) HLX gene was obtained from human umbilical cord blood mononuclear cells by RT-PCR method, cloned into pMD19-T vector, transformed into E.coli DH5 偽 host bacteria, and identified by enzyme digestion. The positive clones were further sequenced. (2) the whole ORF region of HLX gene was obtained from HLX positive clone by PCR, ligated to the prokaryotic expression vector of pQE30, transformed into E.coli DH5 偽 host strain, digested by enzyme and sequenced. The positive recombinant plasmid pQE30-HLX was transformed into E.coli M15 strain and was induced to express by isopropyl 尾 -D-thiogalactopyranoside pQE30-HLX. The expressed HLX fusion protein was identified by SDS-PAGE electrophoresis and Western blot. A large number of HLX fusion proteins were induced and purified by Profinity IMAC Ni-Charged Resin affinity chromatography. Rabbits were immunized with HLX protein as antigen. Rabbit sera containing polyclonal antibodies against HLX were collected. The titers and specificity of the antibodies were identified by ELISA and Westernblot methods. The expression of HLX in peripheral blood mononuclear cells (PBMC) of patients with gastric cancer was detected by quantitative PCR and compared with that of healthy controls. The relationship between the expression of HLX and the expression of T-beta-GATA-3 in gastric cancer patients was also analyzed. In order to understand the imbalance of Th1/Th2 in patients with gastric cancer from the transcriptional factor level. Results: The human HLX gene was cloned successfully and the pMD19-T-HLX plasmid was constructed. The ORF region of human HLX gene was cloned successfully and the prokaryotic expression plasmid of pQE30-HLX was constructed. The result of sequencing showed that the target fragment ligated to the pQE30 vector was the ORF region of HLX, with a length of 1467bp, encoding 488 amino acid residues, which was identical to the sequence published in GenBank. HLX fusion protein was obtained by induction and purification, and the size of the fusion protein was about 55 KD. The polyclonal antibody against human HLX protein was successfully prepared, which was identified by ELISA and Western blot and showed good reactivity and specificity. It also showed that HLX fusion protein had good immunogenicity. The expression of PBMC HLX in patients with gastric cancer was detected by real-time fluorescence quantitative PCR, which was lower than that in healthy controls. The expression of T-bett GATA-3 was detected by the same method in patients with gastric cancer, and the correlation between the two genes was analyzed. Conclusion: The prokaryotic expression plasmid of pQE30-HLX was successfully constructed and effectively expressed in Escherichia coli. The fusion protein of human HLX was obtained and used as antigen to prepare specific polyclonal antibody. The expression level of HLX in PBMC of gastric cancer patients was detected. Compared with the healthy control group, the expression level was positively correlated with other Th1 type cytokines or transcription factor T-bet, and negatively correlated with GATA-3. It may be related to the occurrence and development of gastric cancer.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392.11

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本文編號(hào)：1812756