大鼠T细胞受体Vβ5.2-HSP70和Vβ8.2-HSP70基因真核表达质粒的构建及表达

Construction and expression of eukaryotic expression vectors containing rat T-cell receptor V beta 5.2-HSP70 and V beta 8.2-HSP70 genes

目的:构建含大鼠TCR Vβ5.2-HSP70和TCR Vβ8.2-HSP70基因的重组真核表达质粒,并检测其在体内外的表达。方法:实验于2005-10/2006-09在首都医科大学免疫学系实验室完成。①以自主构建的重组表达载体pTARGET-TCR Vβ5.2和pTARGET-TCR Vβ8.2为模板,将结核杆菌HSP70的一段保守序列P111-125设计到引物中,通过PCR的方法得到TCR Vβ5.2-HSP70和TCR Vβ8.2-HSP70基因片段,将其插入到真核表达载体pTARGET中,构建重组表达载体pTARGET-TCR Vβ5.2/8.2-HSP70。②将BALB/c小鼠48只随机分为4组,pTARGET-TCR Vβ 5.2-HSP70组、pTARGET-TCR Vβ8.2-HSP70组和空质粒组肌注相应的质粒DNA100μg/次,PBS组注射PBS缓冲液100μL/次。共注射3次,每次间隔2周。于末次免疫后3d取注射部位的肌肉,用RT-PCR及免疫组化染色法检测TCR Vβ5.2/8.2-HSP70基因在注射部位的转录和表达。③将COS-7细胞分为pTARGET-TCR Vβ5.2-HSP70组、pTARGET-TCR Vβ8.2-HSP70组、空质粒组和未转染细胞组,进行相应质粒转染,24h后收集细胞,用免疫细胞化学染色法检测靶基因在真核细胞内的表达。结果:①TCR Vβ5.2/8.2-HSP70基因已被正确插入到pTARGET载体中。②pTARGET-TCR Vβ5.2-HSP70组、pTARGET-TCR Vβ8.2-HSP70组小鼠免疫处的肌肉组织RT-PCR检测显示,在500～600bp和400～500bp处出现特异的扩增带,分别与TCR Vβ5.2-HSP70和TCR Vβ8.2-HSP70基因片段的大小相符,而注射空质粒组和PBS组均未见此条带。③免疫组化结果显示转染重组质粒pTARGET-TCR Vβ5.2/8.2-HSP70的COS-7细胞的胞质及胞膜都有明显的着染。结论:成功构建了重组真核表达质粒pTARGET-TCR Vβ5.2/8.2-HSP70,并在体内外检测到其转录和表达,为进一步进行TCR DNA疫苗治疗胶原诱导性关节炎的实验研究奠定了基础。

AIM：To construct the recombinant eukaryoUc expression vector pTARGET-TCR Vβ 5.2/8.2-HSP70 and detect their expressions. METHODS： The experiment was performed at the Laboratory of Department of Immunology, Capital Medical University from October 2005 to September 2006. ①Gene encoding TCR Vβ 5.2-HSP70 was amplified by PCR from recombinant plasmid pTARGET-TCR Vβ 5.2 and TCR Vβ 8.2-HSP70 was from recombinant plasmid pTARGET-TCR Vβ 8.2. Then they were cloned into eukaryotic expression vector pTARGET.②Totally 48 BALB/c mice were randomly divided into 4 groups： pTARGET-TCR Vβ 5.2-HSP70 group, pTARGET- TCR Vβ 8.2-HSP70 group, plasmid group （injecting DNA intramuscularly once 100 μg） and PBS group （injecting PBS buffer once 100μL）, totally injecting for 3 times, interval for 2 weeks once. The injected skeletal muscles were isolated 3 days after the last immunity, and the transcription and expressions of TCR Vβ 5.2/8.2-HSP70 genes were detected by RT-PCR and immunohistochemical staining. ③COS-7 cells were assigned into pTARGET-TCR Vβ 5.2-HSP70 group, pTARGET-TCR Vβ 8.2-HSP70 group, plasmid group and non-transfecUon group. Corresponding plasmid transfection was. conducted, and cells were collected 24 hours later. Expression of target gene in eukaryotic cells was measured with immunocytochemical staining. RESULTS： ①TCR Vβ5 5.2/8.2-HSP70 genes were successfully inserted into pTARGET. ②RT-PCR indicated that special amplified band appeared at 500-600 bp and 400-500 bp in the injected muscles, which were accorded with the size of TCR Vβ 5.2-HSP70 and TCR Vβ 8.2-HSP70 gene segments in the pTARGET-TCR Vβ 5.2-HSP70 group and pTARGET. TCR Vβ 8.2-HSP70 group. But there were no band in the plasmid group and PBS group. ③ Immunohistochemical staining showed there was significant staining in the kytoplasm and cell membrane of COS-7 cells of pTARGET-TCR Vβ 5.2/8.2-HSP70. CONCLUSION： Theeukaryotic expression vector pTARGET-TCR Vβ 5.2/8.2-HSP70 are successfully constructed and expressed in vivo and vitro, which will lay foundation on the protective effects of TCR DNA vaccine on collagen induced arthritis （CIA）.