红螯螯虾卵黄蛋白原N端结构域的生物信息学分析与蛋白的表达鉴定  

Bioinformatics analysis and protein expression identification of vitellogenin N-terminal domain in Cherax quadricarinatus

作　　者：黄立斌 武霞 王瑞 周明瑞 黄光华 黄黎明 吕敏 黄峥 杨慧赞 HUANG Libin;WU Xia;WANG Rui;ZHOU Mingrui;HUANG Guanghua;HUANG Liming;LYU Min;HUANG Zheng;YANG Huizan(Guangxi Academy of Fishery Sciences,Guangxi Key Laboratory of Aquatic Genetic Breeding and Healthy Aquaculture,Nanning 530021,China;College of life sciences,Guangxi Normal University,Guilin 541006,China)

机构地区：[1]广西壮族自治区水产科学研究院广西水产遗传育种与健康养殖重点实验室,南宁530021 [2]广西师范大学生命科学学院,广西桂林541006

出　　处：《黑龙江畜牧兽医》2022年第10期120-127,136,137,共10页Heilongjiang Animal Science And veterinary Medicine

基　　金：广西科技重大专项课题(桂科AA17204095-5,桂科AA20302019-4);广西自然科学基金项目(2019GXNSFAA185036);广西重点研发计划项目(桂科AB18221068)。

摘　　要：为研究红螯螯虾(Cherax quadricarinatus)卵黄蛋白原N端结构域(vitellogenin N domain,VitN)的生物学功能并获得具有免疫特异性的红螯螯虾VitN融合蛋白,试验根据红螯螯虾卵黄蛋白原的mRNA序列(GenBank登录号为AF306784.1)获得VitN mRNA序列(编码第42~585位氨基酸),通过在线分析工具对红螯螯虾VitN进行生物信息学分析,并优化合成红螯螯虾VitN基因序列,构建重组表达质粒pET-28a-VitN,将其转化至大肠杆菌BL21(λDE3)感受态细胞中,经IPTG诱导,对红螯螯虾VitN融合蛋白进行表达、纯化、包涵体复性、Western-blot鉴定和浓度测定。结果表明:编码红螯螯虾VitN的氨基酸共544个,均为常见氨基酸,其中丙氨酸、缬氨酸和丝氨酸含量较多(占比分别为7.72%、7.72%和7.54%),色氨酸含量最少(仅占0.92%);红螯螯虾VitN的相对分子质量为64681,理论等电点为8.69,共含有8540个原子,分子式为C_(2671)H_(4260)N_(758)O_(818)S_(33),不稳定系数为31.49,脂肪系数为74.56,亲水性平均值为-0.45,亲水氨基酸数量明显多于疏水氨基酸数量;编码红螯螯虾VitN的氨基酸全部位于细胞膜外,不存在跨膜区;红螯螯虾VitN亚细胞定位在细胞外或细胞质内,不含常规信号肽,也不含Ⅰ型信号肽酶;红螯螯虾VitN共有67个磷酸化位点,其中丝氨酸磷酸化位点31个,苏氨酸磷酸化位点23个,酪氨酸磷酸化位点13个;红螯螯虾VitN的二级结构包括α-螺旋、β折叠、延伸链、无规则卷曲;三级结构包含具有脂质结合功能的lv-1n和lv-1c肽链结构,且呈现上下两个较为明显的区域,上半部分为sheet折叠结构富集区域,下半部分为α螺旋结构富集区域;在红螯螯虾第129~153位氨基酸和第396~402位氨基酸处各存在一个二硫键。优化后的红螯螯虾VitN基因片段大小约为1650 bp;经连接、转化、诱导,获得了大小约为64.7 ku的融合蛋白,其在大肠杆菌BL21(λDE3)感受态细胞中的主要表达形式为包涵体;最佳诱To study the biological functions of vitellogenin N domain(VitN)in Cherax quadricarinatus,and obtain VitN fusion protein with immune specificity,VitN mRNA sequence(coding 42-585 aa)was obtained according to the mRNA sequence of vitellogenin of Cherax quadricarinatus(GenBank accession number AF306784.1).Bioinformatics analysis of VitN was carried out by online analysis tools.VitN gene sequence was optimized and synthesized to construct recombinant expression plasmid pET-28a-VitN.The fusion protein was transformed into Escherichia coli BL21(λDE3)cells and induced by IPTG.The expression,purification,inclusion body renaturation,Western-blot identification and concentration determination of VitN fusion protein were performed.The results showed that there were 544 amino acids encoding VitN,all of which were common amino acids,among which alanine,valine and serine were the most,accounting for 7.72%,7.72%and 7.54%,respectively.Tryptophan content was the least,accounting for only 0.92%.The relative molecular weight of VitN was 64681;the theoretical isoelectric point was 8.69,a total of 8540 atoms;the molecular formula was C_(2671)H_(4260)N_(758)O_(818)S_(33),the instability coefficient was 31.49;the fat coefficient was 74.56,and the average hydrophilic value was-0.45.The number of hydrophilic amino acids was significantly higher than that of hydrophobic amino acids.The amino acids encoding VitN were all located outside the cell membrane without transmembrane region.VitN subcellular localization was extracellular or cytoplasmic examination.It did not contain conventional signal peptides,nor did it contain typeⅠsignal peptidase.There were 67 phosphorylation sites in VitN,including 31 serine phosphorylation sites,23 threonine phosphorylation sites,and 13 tyrosine phosphorylation sites.The secondary structure of VitN consisted of α-helixes,βfolds,extended chains,and random curls.VitN contained lipid-binding lv-1n and lv-1c peptide chains.There were two obvious regions in the upper and lower parts of the tertiary structur

关 键 词：卵黄蛋白原 N端结构域 生物信息学分析 原核表达 分离纯化 

分 类 号：S966.12[农业科学—水产养殖]