T淋巴瘤TCR V_β核酸疫苗的构建和生物学活性的初步检测

Construction of TCR V_β Nucleic Acid Vaccine and Testing on Its Biological Effects

目的：制备Ｔ淋巴瘤ＴＣＲＶβ核酸疫苗。方法：利用ＲＴ－ＰＣＲ方法扩增出人Ｔ淋巴瘤细胞Ｊｕｒｋａｔ的ＴＣＲ Ｖβ基因片段，并将其重组入真核表达载体ｐｃＤＮＡ３。用重组表达载体转染ＳＰ２／０细胞，在ｍＲＮＡ水平上检测其表达。再用ｐｃＤＮＡ３和重组载体分别免疫ＢＡＬＢ／ｃ小鼠，收集抗血清，用免疫组化技术检测抗体产生情况。结果：扩增出ＴＣＲ Ｖβ的基因片段，测序结果证实其序列与已发表的一致。并构建出重组表达载体ｐｃＤＮＡ３－ＴＣＲ Ｖβ。该质粒转染到 ＳＰ２／０细胞，可在ｍＲＮＡ水平上检测到其表达。在用ｐｃＤＮＡ３－ＴＣＲＶβ免疫的小鼠抗血清中检测到抗Ｊｕｒｋａｔ细胞 ＴＣＲ Ｖβ的抗体。免疫组化结果表明，该血清不与表达 ＴＣＲγδ的人Ｔ淋巴瘤细胞发生反应，而与转染该基因的ＳＰ２／０细胞产生强阳性反应。正常小鼠血清与ｐｃＤＮＡ３免疫的小鼠血清与Ｊｕｒｋａｔ细胞不产生显色反应。结论：所制备的Ｔ淋巴瘤ＴＣＶβ核酸疫苗能有效地诱导机体产生体液兔疫反应。

Objective: To construct TCR Vβ nucleic acid vaccine. Methods: A fragment of TCR Vβ gene was amplified by RT- PCR from a human T lymphoma cell line, Jurkat, and cloned into eukaryotic expression plasmid pcDNA3 by gene recombination. After sequencing, the recombinant plasmid was transfected into SP2/0 cells and its expression was detected on mRNA level. BALB/c mice were immunized with pcDNA3 and the recombinant plasmid by intramuscular routes. Antiserum was collected and detected by immunocytochemistry method. Results: A fragment of TCR Vβ gene from Jurkat cells was obtained and proved to be identical to published sequence. A recombinant plasmid pcDNA3-TCR Vβ was comstructed. Its expression was detectable on mR- NA level after being transfected into SP2/0 cells. Antiserum from mice immunized with pcDNA3-TCR Vβ reacted strongly with Ju- rkat cells and SP2/0 cells transfected by pcDNA3-TCR Vβ, while shows no reaction on CEM cells expressing TCRγδ and SP2/0 cells. Antisera from normal force and mice immunized with pcDNA3 were both negative on Jurkat cells. The results of immunocy- tochemistry indicated that BALB/c mice immunized with pcDNA3-TCR Vβ produced specific antibody to Jurkat TCR Vβ.Conclu sion: The TCR Vβ nucleic acid vaccine we constructed can induce humoral immunity.