摘要
目的研究IgG Fc编码基因对流行性乙型脑炎（Japanese encephalitis，JE）DNA疫苗免疫增强效应的影响。方法巢式RT-PCR法从BALB／c鼠脾组织获取IgG Fc段编码基因，用限制性内切酶从含流行性乙型脑炎病毒（Japanese encephalitis virus，JEV）prME蛋白基因重组子获取prME蛋白基因，分别插入同一真核表达载体pcDNA3．1（＋）不同酶切位点，构建重组子pJME／IgG Fc并经酶切及DNA测序分析。脂质体法将pJME／IgG Fc转染CHO细胞。免疫荧光、Western blot法检测转染的CHO细胞中融合蛋白分布与表达。将pJME／IgG Fc肌注免疫BALB／c鼠，检测小鼠脾特异性细胞毒T细胞（CTL）杀伤活性和中和抗体滴度。结果pJME／IgGFc经BamHI／EcoRI和BamHI／NotI酶切释出的插入子大小（2001 bp、2730 bp）分别与预期结果相符合。所编码的融合蛋白相对分子质量（Mr）为101×10^3，主要分布于胞浆，少量分布于胞膜，pJME／IgG Fc转染CHO细胞经32次传代仍可表达融合蛋白。pJME／IgG Fc免疫组中和抗体滴度与CTL活性较pJME及灭活疫苗组均升高（P〈0．05）。结论pJME／IgG Fc成功构建，转染的CHO细胞可稳定表达融合蛋白，IgG Fc段编码基因能够增强JEV DNA疫苗的细胞和体液免疫应答。
Objective To study the effect of IgG Fc gene on JEV DNA vaccine immunity. Methods Gene encoding IgG Fc was amplified by nested-RT-PCR technique from BALB/c murine spleen cells. JEV prME protein gene was obtained with restriction endonuclease BamH I/EcoR I from the eukaryotic recombinant named after pJME, which was constructed by us before. Recombinant, named after pJME/IgG Fc, with above two genes encoding JEV prME protein and BALB/c murine IgG Fc was constructed, and was tested by restriction enzymes analysis and DNA sequencing, then was transfected into China hamster ovary （CHO） cells by LipofectAMINE 2000. Distribution and expression of the fusion proteins encoded by JEV prME protein and BALB/c murine IgG Fc genes in transfected CHO cells were detected by immunofluorescence and Western blot. The BALB/c micc were vaccinated with pJME/IgG Fc via intramuscular injection. Then the cytotoxic T lymphocyte （CTL） activity were assessed by lactic dehydrogenase （LDH） and the neutralizing antibody titer were assessed by 80% plaque reduction neutralization test. Results Molecular weights （2001 bp, 2730 bp） of the two inserts released from pJME/IgG Fc with two group of restriction analysis associated with BamH I/EcoR I and BamH I/Not I were correlated to the expected theoretic results respectively. It was estimated that molecular weight （Mr） of the fusion protein was 101 x 103. The expression of the above fusion protein was mainly distributed in endochylema of transfected CHO cells, and not much in membrane of transfected CHO cells. CHO cells transfected with pJME/IgG Fc could express the fusion protein at the 32th cell passage. After immunization, the CTL activity and the neutralizing antibody titer in the pJME/IgG Fc vaccinated group increased significantly compared with other vaccinated groups（P 〈0.05）. Conclusion The recombinant pJME/IgG Fc was constructed and transfected into CHO cells successfully, and CHO cellular lines expressed fusion protein encoded by JEV prME protein and BALB/c murine
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2008年第7期634-638,共5页
Chinese Journal of Microbiology and Immunology
基金
基金项目：国家自然科学基金（30471547）
教育部留学回国人员启动基金（教外司留[2004]527）
辽宁省自然科学基金（20042072）
辽宁省教育厅重大基础研究计划（05L498）
