小鼠BTLA胞外功能区基因克隆表达及其对DC表面B7分子表达的影响

目的:研究重组谷胱甘肽S-转移酶(GST)-小鼠B/T细胞弱化因子包外功能区(mBTLAext)融合蛋白(GST-mBT-LAext)对小鼠树突状细胞系DC2.4表面B7分子表达的影响。方法:采用RT-PCR技术从小鼠脾细胞总RNA中逆转录mB-TLAcDNA。将其胞外功能区基因克隆入原核表达载体pGEX-4T-2中,构建重组表达质粒pGEX-4T-2/mBTLAext并转化E.coliBL21(DE3),以1mmol/L的IPTG诱导表达。提取包涵体,经变性、负性后,采用GlutathioneSepharose4B柱纯化可溶性的GST-mBTLAext。将不同浓度的GST-mBTLAext加入到DC2.4的培养体系中,采用流式细胞术检测其对DC上B7-1和B7-2表达的影响。结果:成功地克隆了mBTLA基因,并构建了重组原核表达载体。变性、复性,经GlutathioneSepharose4B柱纯化,获得了可溶性的GST-mBTLAext融合蛋白。经SDS-PAGE鉴定表明,融合蛋白的相对分子质量(Mr)为43000,同预期的结果一致。流式细胞术检测显示,GST-mBTLAext可上调DC2.4上B7-1的表达并呈剂量依赖性,这一作用可被抗GST-mBTLAext血清阻断。未检测到GST-mBT-LAextDC2.4上B7-2的表达有影响。结论:BTLA对DC2.4表面B7分子表达的上调可能是BTLA-HVEM途径反向信号对DC作用的结果,对进一步研究其对DC生物学行为的影响及其分子机制具有重要的理论意义。

Clone and expression of murine BTLA extracellular domain gene and its effect on the expression of B7 on dendritic cells

AIM: To study the regulatory effect of recombinant glutathione S-transferase (GST)-extracellular domain of murine B and T lymphocyte attenuator (mBTLAext) fusion protein GST-mBTLAext on the expression of B7 on murine dendritic cell (DC) line DC2.4. METHODS: cDNA of mBTLA was amplified from total RNA of murine splenocytes by RT-PCR. The recombinant prokaryotic expression vector pGEX-4T-2/mBTLAext was constructed by cloning mBTLAext into pGEX-4T-2 and then transformed into E. coli BL21 (DE3). The fusion protein GST-mBTLAext was expressed under the induction of 1 mmol/L IPTG, and then was extracted from inclusion body and purified through Glutathione Sepharose 4B chromatography column. The fusion protein was added into the culture supernatant of DC2.4 and its effect on the expression of B7-1 and B7-2 on DC2.4 was analyzed by flow cytometry. RESULTS: The recombinant prokaryotic expression vector pGEX-4T-2/mBTLAext was constructed and the fusion protein GST-mBTLAext was expressed successfully. The molecular weight of the fusion protein was 43.0 kDa, determined by SDS-PAGE, which was corresponding to expectation. GST-mBTLAext could up-regulate the expression of B7-1, but didn't alter the expression of B7-2, on DC2.4 in a dose dependent manner. CONCLUSION: BTLA had a regulatory effect on the expression of B7 on DC. It is significant to study the effect of BTLA on the biological behaviour of DC and its molecular mechanism.