| 猪恒定链的原核表达及其分子结构与功能分析 |
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| 引用本文： | 董林,王艳萍,吕素芳,王金良,唐娜,沈志强.猪恒定链的原核表达及其分子结构与功能分析[J].动物医学进展,2016(10):64-69. |
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| 作者姓名： | 董林 王艳萍 吕素芳 王金良 唐娜 沈志强 |
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| 作者单位： | 1. 山东省滨州畜牧兽医研究院，山东滨州 256600; 山东绿都生物科技有限公司，山东滨州 256600;2. 山东省滨州畜牧兽医研究院,山东滨州,256600 |
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| 基金项目： | 山东省自然科学基金项目(ZR2013CQ006) |
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| 摘 要： | 为研究猪恒定链(Ii)分子结构与功能,通过PCR扩增编码完整Ii及其异构体的基因片段,克隆至T pMD18-T载体,在对目的基因进行测序及分析其基因与编码蛋白分子结构,确定功能域及其分子遗传变异的基础上,构建pET-32a-Ii重组载体,转化BL21进行诱导表达,并分析表达产物的免疫活性。结果显示,PCR扩增了645bp和837bp的Ii及其异构体编码基因,DNA序列分析其编码氨基酸分别为215个和279个,异构体Ii多出了64个氨基酸构成的Tg区;分子结构分析显示,猪Ii分子由胞内区、跨膜区和胞外区3部分组成,包含有内体定位基元、Ii-key、CLIP、TRIM等功能域,其中内体定位基元含有特征性L7I和PML17双亮氨酸基序,Ii-key、CLIP、TRIM氨基酸序列与人和小鼠Ii具有高度一致性。遗传变异分析表明,猪Ii分子与人、小鼠等哺乳动物的Ii同源性为62.7%~95.6%,与鸡、鸭、鸽、鹅等禽类Ii的同源性为48.7%~49.5%;空间结构分析显示,猪Ii有3个相似的α-helix构成,其间间隔由相似氨基酸构成的2个转角,转角关键位点氨基酸具有高度一致性;Ii编码基因在BL21细胞获得了有效表达,目的蛋白大小约41ku,Western blot和免疫荧光检测显示,表达产物具有良好的反应原性。试验成功获得了猪Ii及其异构体编码基因,明确了其功能区、分子遗传变异等特征,诱导表达获得了具有良好免疫活性的Ii重组蛋白,为进一步研究猪Ii分子结构与功能奠定了基础。
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| 关 键 词： | 猪 恒定链 原核表达 分子结构 功能分析 |
Prokaryotic Expression of Porcine Ii and Analysis of Its Molecular Structure and Function |
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| Abstract: | The molecular structure and function of porcine Ii were researched by PCR amplification of Ii and its isomers conding cDNA,which the gene fragment was cloned into T-Vector for DNA sequence.The gene and encoding protein molecular structure were analyzed and the functional domain and its molecular genetic variation were detemined.The expression vector of pET-32a-Ii was constructed and transformed into BL21 that was induced for protein expression,and the immune activity of protein was analyzed.The results showed that the complete genes of porcine invariant chain and its isomer were 645 nt and 837 nt respective-ly,each coding 215 aa and 279 aa.Compared to the invariant chain,the isomer had an extra Tg domain enco-ded by 64 aa.Molecular structure analysis predicted that the porcine invariant chain had intracellular, transmembrane and extracellular zones which included several function domains such as inner body positio-ning element,Ii-key,CLIP and TRIM.The inner body positioning element had distinctive dual leucine mo-tifs L7I and PML17,and all the amino acid sequences of Ii-key,CLIP,TRIM showed high consistencies with human and mouse Ii.The genetic variation analysis illustrated that porcine invariant chain was 62.7%-95.6% homologous to human or mouse invariant chain,and 48.7%-49.5% to chicken,duck,dove, goose and other birds.The porcine invariant chain was composed of three similarα-helixes,spaced with two corners which had highly similar amino acids.The 41 ku recombinant protein was also effectually expressed in BL21 cells,showed good reactivity in Western blot and IFA tests.The present study will provide useful information for the further study of porcine invariant chain molecular structure and function. |
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| Keywords: | pig invariant chain prokaryotic expression molecular structure function analysis |
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