CAJ | 학술논문

目的研究人B7.2分子中IgV和IgC 结构域何者含有与受体结合的位点；对由原核系统表达的B7.2蛋白体外共刺激活性作出评价。方法　用PCR方法扩增人B7.2分子胞外区的三个片段，即IgV、IgC和包括IgV和IgC在内的Ig（V+C）区段，将三片段克隆入pGEM-Teasy。继而将三片段克隆入原核表达载体pGEX-4T-3，得到pGEX-4T-3-hB7.2 IgV、pGEX-4T-3-hB7.2 IgC和pGEX-4T-3-hB7.2 Ig(V+C)三个重组体，将三重组体转化宿主菌DH5α。经SDS-PAGE和Western blot证实与GST融合存在的相应靶蛋白的表达。用抗CD3单抗模拟T细胞活化的第一信号，用［3H］-TdR掺入法观察加入目的蛋白作为第二信号可否对T细胞活化起协同效应。结果　工程菌可表达出人B7.2分子的三种融合蛋白：GST-hB7.2 IgV、GST-hB7.2 IgC和GST-hB7.2 Ig(V+C)，且以包涵体形式存在。在第一信号存在条件下，GST-hB7.2 Ig(V+C)和GST-hB7.2 IgV均能协同第一信号活化T淋巴细胞，但GST-hB7.2 IgC无此功能。结论　细菌可表达出具有功能活性的人B7.2融合蛋白，单一的人B7.2 IgV结构域足以与受体结合，共刺激T细胞活化。
목적연구인B7.2분자중IgV화IgC 결구역하자함유여수체결합적위점；대유원핵계통표체적B7.2단백체외공자격활성작출평개。 방법　용PCR방법확증인B7.2분자포외구적삼개편단，즉IgV、IgC화포괄IgV화IgC재내적Ig（V+C）구단，장삼편단극륭입pGEM-Teasy。계이장삼편단극륭입원핵표체재체pGEX-4T-3，득도pGEX-4T-3-hB7.2 IgV、pGEX-4T-3-hB7.2 IgC화pGEX-4T-3-hB7.2 Ig(V+C)삼개중조체，장삼중조체전화숙주균DH5α。경SDS-PAGE화Western blot증실여GST융합존재적상응파단백적표체。용항CD3단항모의T세포활화적제일신호，용［3H］-TdR참입법관찰가입목적단백작위제이신호가부대T세포활화기협동효응。결과　공정균가표체출인B7.2분자적삼충융합단백：GST-hB7.2 IgV、GST-hB7.2 IgC화GST-hB7.2 Ig(V+C)， 차이포함체형식존재。재제일신호존재조건하，GST-hB7.2 Ig(V+C)화GST-hB7.2 IgV균능협동제일신호활화T림파세포，단GST-hB7.2 IgC무차공능。 결론　세균가표체출구유공능활성적인B7.2융합단백，단일적인B7.2 IgV결구역족이여수체결합，공자격T세포활화。
Objective To investigate which of the two immunoglobulin (Ig)-like domains, the immunoglobulin variable region homologous domain IgV (hB7.2 IgV) and the immunoglobulin constant region homologous domain IgC (hB7.2 IgC) on the human B7.2 molecule contains receptor binding sites, and to evaluate whether the B7.2 protein expressed in bacteria has biological activity in vitro. Methods Three fragments of hB7.2 IgV, hB7.2 IgC and the complete extracellular region of human B7.2 containing both the IgV and IgC domains, hB7.2 Ig (V+C), were amplified by PCR and subcloned into pGEM-Teasy. Three recombinants, pGEX-4T-3-hB7.2 IgV, pGEX-4T-3-hB7.2 IgC and pGEX-4T-3-hB7.2 Ig (V+C), were generated by cloning the fragments into a prokaryote expression plasmid (pGEX-4T-3) and transformed into the host strain E. Coli DH5α. The relevant target fusion proteins consisting of GST and hB7.2 IgV, hB7.2 IgC and hB7.2 Ig (V+C), were identified by SDS-PAGE and Western blotting. With the presence of the first signal imitated by anti-CD3 antibody, T cell activation was observed by exposing purified T lymphocytes to each soluble form of the three bacterially-produced human B7.2 fusion proteins by [3H]-TdR incorporation. Results Three recombinant fusion proteins of human B7.2, GST-hB7.2 IgV, GST-hB7.2 IgC and GST-hB7.2 Ig (V+C) were produced and detected in inclusion body form from engineered bacteria. With the first signal present, T lymphocytes proliferated when co-stimulated by bacterially-produced either GST-hB7.2 Ig (V+C) or GST-hB7.2 IgV fusion proteins, but not by GST-hB7.2 IgC.Conclusions Functional human B7.2 fusion protein can be produced in bacteria. The IgV-like domain of human B7.2 is sufficient for B7.2 to interact with its counter-receptors and co-stimulate T lymphocytes.