摘要
目的:了解金黄色葡萄球菌肠毒素A(SEA)联合PML-RARα融合多肽体外诱导正常人外周血T细胞活化TCRζ链基因表达情况;分别构建SEA基因全长的真核表达质粒和BCR-ABL融合基因的真核表达质粒,并检测它们在真核细胞中的表达情况,为进一步联合SEA基因的超抗原性研制白血病疫苗提供可行性资料和前期性研究数据。
方法:1,利用T细胞液体培养法分别于正常人外周血淋巴细胞加入PML-RARα融合多肽、SEA和SEA联合PML-RARα多肽诱导培养T细胞;2,采用SYBR GreenⅠ荧光定量PCR和相对定量检测TCRζ链在不同组别T淋巴细胞中的表达情况,以β2微球蛋白基因(β2M)作为内参,根据相对定量公式:2~(-△△Ct)分析TCRζ链表达差异;3,利用RT-PCR技术从K562细胞中扩增出BCR-ABL基因片段,从金黄色葡萄球菌菌株基因组DNA中PCR扩增SEA基因全长,应用分子克隆技术,分别克隆到pIRES2-EGFP真核表达载体上,构建pIRES2-EGFP-BCR-ABL和pIRES2-EGFP-SEA真核表达质粒。两重组质粒经多种分子生物学方法进行鉴定后进行基因测序;4,重组质粒利用Lipofectamine~(TM)2000转染K293细胞,荧光显微镜下拍照并RT-PCR技术检测转录水平的表达情况。
结果:1,与空白组相比,联合诱导组在培养初始加入SEA及第5天加入SEA的培养T细胞中TCRζ链表达上升,而单独SEA诱导组的TCRζ链表达下降。2,所构建的pIRES2-EGFP-BCR-ABL和pIRES2-EGFP-SEA真核表达质粒通过PCR可克隆得到相应大小的产物;酶切鉴定所切下的两片段大小约为342bp以及813bp,与预计相符;测序结果与报道一致。3,两重组质粒转染K293细胞后,RT-PCR方法检测出外源基因BCR-ABL与SEA与预计相符,并荧光显微镜下可见绿色荧光蛋白表达,
结论:1,超抗原SEA联合PML-RARα多肽体外诱导T细胞可使TCRζ链基因表达水平升高,有望为研制急性早幼粒细胞白血病疫苗提供新的切入点;2,成功地从k562细胞株中扩增出目的基因BCR-ABL并构建了重组真核表达载体pIRES2-EGFP-BCR-ABL;3,成功地从金黄色葡萄球菌菌株中扩增出目的基因SEA并构建了重组真核表达载体pIRES2-EGFP-SEA;4,这些重组质粒在体外转染真核细胞后,插入到重组质粒中的外源基因能够在真核细胞K293中正常地转录并表达绿色荧光蛋白。
Objective:To measure the expression of signaling transduction factor T lymphocyte-TCR chain gene in peripheral blood mononuclear cells which stimulated by PML-RARαfusion peptide or SEA or both of them.To construct the eukaryotic expression plasmid of BCR-ABL gene and SEA gene respectively.
Methods:1,Peripheral blood mononuclear cells from normal individual were cultured with PML-RARαpeptide,SEA and both of them,which were induced by PML-RARαpeptide for 20 days,respectively add the SEA at the beginning,the fifth day,and a comparison of SEA was set.2,Real-Time PCR with SYBR GreenⅠtechnique was used for detecting TCRζchain expression level andβ2-microglobulin gene(β2M) was used as an endogenous reference.Relative changes in TCRζchain expression level were used by the 2~(-ΔΔCt) method between each group and the control.3,BCR-ABL fusion gene segments were amplified from K562 leukemia cell by RT-PCR and were inserted into the MCS A of pIRES2-EGFP plasmid to construct a recombinant plasmid pIRES2-EGFP-BCR-ABL.The whole SEA gene were amplified from the DNA of staphyloccocus aureus by PCR and to construct a recombinant plasmid pIRES2-EGFP-SEA.4,To transfect the pIRES2-EGFP-BCR-ABL and the pIRES2-EGFP-SEA into K293 cells respectively,detect the protein expression of EGFP,and to detect the mRNA expression of BCR-ABL genes and SEA genes by RT-PCR.
Results:1,The expression level of TCRζchain in groups which were added the SEA at the beginning and the fifth day were over expressed,the other groups were decreased;2,The results of PCR,double restriction endonuclease cutting and DNA sequencing analysis confirmed that the pIRES2-EGFP-BCR-ABL and the pIRES2-EGFP-SEA were successfully constructed;3,Both EGFP protein and mRNA expressed by recombinant plasmids in K293 cell could be correctlly detected.
Conclusion:1,The expression of signaling transduction factor T lymphocyte-TCRζchain gene in peripheral blood mononuclear cells,which stimulated by SEA and PML-RARαfusion peptide,could be over expressed.2,Successfully construct the recombinant plasmid of the plRES2-EGFP-BCR-ABL 3,Successfully construct the recombinant plasmid of the pIRES2-EGFP-SEA 4,These plasmids have normal function of transcription and can express the protein of EGFP in eukaryotic cells in vitro.
引文
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