构建免疫球蛋白与细胞因子融合基因表达载体的家族特异性抗淋巴瘤疫苗

Development of IgHV family specific nucleic acid vaccine against lymphoma by construction of fusion gene of immunoglobulin heavy chain variable region and cytokine

目的 构建免疫球蛋白重链可变区 (IgHV)基因片段与细胞因子粒细胞 巨噬细胞集落刺激因子 (GM CSF)或白细胞介素 2 (IL 2 )的融合基因表达载体作为家族特异性抗淋巴瘤核酸疫苗 ,接种动物了解该疫苗抗淋巴瘤免疫功能。方法 从脐带血免疫球蛋白基因文库获得 6个片段长度差异较大的IgHV3基因片段 ,测序后利用生物信息资源分析预测其重链可变区的T细胞表位 ,分析IgHV1 6个片段。选择包含了绝大多数T细胞表位的IgHV1、IgHV3基因 ,将框架区 (FR)为主的IgHV(FR)基因片段及IgHV(FR)与GM CSF或IL 2基因连接后形成的融合基因克隆到真核表达载体中。表达载体通过脂质体转染COS细胞 ,ELISA法确定GM CSF或IL 2的表达情况。将一些表达载体作为核酸疫苗免疫小鼠 ,通过间接免疫荧光法和细胞因子分泌法检测小鼠抗同一家族淋巴瘤和正常淋巴细胞的免疫反应。结果 计算机评分系统预测显示在每个IgHV序列中约存在 30个左右分别受HLA位点限制的T细胞表位 ,90 %以上的T细胞表位位于框架区 ,积分排位于前 1 0 %的T细胞表位都位于框架区。构建了去除CDR3区后以框架区 (FR)为主的IgHV1 (FR)和IgHV3(FR)真核表达载体。将GM CSF或IL 2基因连接到 2个IgHV基因的 3′端。IgHV (FR ) GM CSF/pcDNA3.0中GM CSF的表达量高于对照组 2

Objective To construct the gene co expression vector of immunoglobulin heavy chain variable region (IgHV) gene and GM CSF or IL 2 as IgHV family specific nucleic acid vaccine to lymphoma, and study the immune response of the immunized mice against lymphoma Methods Six clones with significantly different length in gene fragments were selected from a gene bank constructed from normal fetal umbilical cord blood These gene fragments in the clones were sequenced The sequences were translated into IgHV peptides T cell epitopes in the IgHV were predicted by bioinformatics Meanwhile 6 clones of IgHV1 constructed before were analyzed The most typical clone of IgHV1 and IgHV3 containing most of the T cell epitopes were selected The IgHV gene fragments whose complementary determining region 3 (CDR3) was cut out and composed mainly of framework region were cloned into eukaryotic expression vector The gene fragments of framework region of IgHV linking with gene of GM CSF or IL 2 were cloned into pcDNA3 0 to form fusion genes of IgHV (FR) GM CSF/IL 2 Then they were transected into COS cells, African green monkey renal cells, by Lipofectin and their transient expression product was detected by ELISA Twenty male Balb/c mice were randomly divided into 5 groups of 4 mice to be injected with different vectors at the weeks 0, 2, and 4: with IgHV3 (FR)/pcDNA3 0, with IgHV3 (FR) IL 2/ pcDNA 3 0, with blank vector pcDNA3 0 as control, with IgHV1 (FR)/pcDNA3 0, and with IgHV1 (FR) IL 2/pcDNA3 0 At the weeks 0, 2, 4, 6, and 8 serum was collected from the mice to undergo indirect immunofluorescence examination to detect the antibodies against Namalwa cells, lymphoma cells from Africa green monkey, and normal lymphocytes ELISA was used to examine the serum interferon (IFN) γ Results About 30 of T cell epitopes existed in each IgHV peptides predicted by bioinformatics Among the bioinformatics prediction, about 90% of the T cell epitopes were in the framework region (FR) of IgHV, and the first 10% with higher prediction score were in FR The CDR3 of two typical IgHV sequences were cut down and the remaining sequences, mainly composed of FR, were used to construct the IgHV (FR)/pcDNA3 0 expression vectors The 3' end of IgHV1 (FR) and IgHV3 (FR) were linked to GM CSF or IL 2 gene successfully The expression of GM CSF in the group transfected with IgHV (FR) GM CSF/pcDNA3 0 were 200 times higher than in the group transfected with control pcDNA3 0 The expression of IL 2 in the group transfected with IgHV (FR) IL 2/ pcDNA3 0 were 60 times higher than in the group transfected with control pcDNA3 0 The antibody against IgHV1 family lymphoma cell line Namalwa could be detected since the second week in the mice immunized with IgHV1 (FR) IL 2/pcDNA3 0 The antibody could be detected since the fourth week in the mice immunized with IgHV1 (FR) The antibody against lymphocyte line of IgHV3 family could be detected since the second week in the mice immunized with IgHV3 (FR) IL 2/pcDNA3 0 The antibody could be detected since the fourth week in the mice immunized with IgHV3 (FR) /pcDNA3 0 The contents of serum IFN γ the mice immunized with IgHV1(FR) IL 2/pcDNA3 0 and with IgHV3(FR) IL 2/pcDNA3 0 was significantly higher than those of the mice immunized with IgHV(FR)/pcDNA3 0 or pcDNA3 0(all P <0 01) Conclusion The gene fragments of IgHV (FR) can be used to construct IgHV family specific nucleic acid vaccine that induces anti lymphoma immune response in mice by muscle injection The expressing vectors of IgHV (FR) GM CSF/IL 2 induces stronger immunoreaction