双峰驼β-NGF基因的克隆及在生殖轴系的表达  

Cloning and expression ofβ-NGF gene in reproductive axis of Bactrian camel

作　　者：王杰 王琪[1,2] 张勇 南京宏[1,2] 成力童 赵兴绪 WANG Jie;WANG Qi;ZHANG Yong;NAN Jinghong;CHENG Litong;ZHAO Xingxu(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;Gansu Provincial Key Laboratory of Animal Reproductive Physiology and Reproductive Regulation,Lanzhou 730070,China)

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]甘肃省动物生殖生理及繁殖调控重点实验室,甘肃兰州730070

出　　处：《中国兽医学报》2022年第11期2242-2249,共8页Chinese Journal of Veterinary Science

基　　金：双峰驼精清诱导排卵的分子调控机理资助项目(31760753);甘肃省动物生殖生理及繁殖调控重点实验室资助项目(20JR10RA563)。

摘　　要：选取健康的雌性双峰驼(5~6岁)的下丘脑、垂体、卵巢、子宫角和输卵管等组织,通过RT-PCR扩增CDS全序列,并进行生物信息学分析;通过qRT-PCR,免疫组织化学染色技术(immunohistochemistry,IHC)和蛋白免疫印记(Western blot)技术检测β-神经生长因子(β-nerve growth factor,β-NGF)在各组织的表达。结果显示,双峰驼β-NGF基因的CDS区(783 bp)可编码261个氨基酸残基,分子式为C_(1219)H_(1914)N_(402)O_(365)S_(11),编码的原子个数为3911,相对分子质量为28.39374 kDa,理论等电点为9.57,β-NGF编码的为非跨膜可溶性蛋白,其二级结构由α-螺旋、延伸链和无规则卷曲构成,该蛋白有22个丝氨酸磷酸化位点,11个苏氨酸磷酸化位点,2个酪氨酸磷酸化位点;同源性分析显示,双峰驼β-NGF的核苷酸序列与单峰驼、羊驼、野猪、山羊、马、黄牛、人、犬、家鼠及家兔的同源性分别为99.6%,99.0%,92.8%,92.6%,91.7%,91.0%,89.5%,89.1%,84.4%,83.7%;IHC结果显示,β-NGF在双峰驼的下丘脑细胞、垂体嗜色细胞和嫌色细胞、卵巢粒细胞、膜细胞和间质细胞、子宫内膜腺上皮细胞和输卵管黏膜上皮细胞均有阳性反应;qRT-PCR结果显示,β-NGF在子宫角中表达量最高,且极显著高于其他组织(P<0.01);Western blot结果显示,β-NGF蛋白在子宫角中表达量最高,且极显著高于下丘脑和输卵管(P<0.01),显著高于垂体(P<0.05)。结果表明,本试验成功克隆出双峰驼β-NGF基因的CDS区,其保守性较高,且该蛋白二级结构有3种折叠方式;β-NGF在子宫角表达水平较高,提示β-NGF可能通过子宫内膜参与了双峰驼的诱导排卵过程。β-nerve growth factor(β-NGF)is an important member of the neurotrophic factor family,which is necessary for the growth,development and repair of various nerve cells and also involved in the ovulation induction of camelidae.In order to explore the physicochemical characteristic ofβ-NGF and its role in the hypothalamic-pituitary-gonad axis of Bactrian camels,the hypothalamus,pituitary,ovary,oviduct and uterus tissues of 5-6 years old healthy female Bactrian camels were selected in this study.The CDS sequence was amplified by RT-PCR and analyzed via bioinformatics.The expression level ofβ-NGF was detected via qRT-PCR,immunohistochemistry(IHC)and Western blot.The CDS region(783 bp)ofβ-NGF gene of bactrian camel was successfully cloned,which encodes 261 amino acids.The molecular formula is C_(1219)H_(1914)N_(402)O_(365)S_(11),encoding 3911 atoms,molecular weight 28.39374 kDa,theoretical isoelectric point is 9.57.β-NGF encodes a non-transmembrane soluble protein,and its secondary structure consists ofα-helix,extended chain and random curl.The protein includes 22serine phosphorylation sites,11threonine phosphorylation sites and 2tyrosine phosphorylation sites.Nucleotide sequence analysis revealed that theβ-NGF gene nucleotide sequence of Bactrian camels was similar to those of dromedary(Camelus dromedarius)(99.6%),alpaca(Vicugna pacos)(99.0%),wild boar(Suss crofa)(92.8%),goat(Capra hircus)(92.6%),horse(Equus caballus)(91.7%),cattle(Bos taurus)(91.0%),mankind(Homo sapiens)(89.5%),dog(Canis lupus familiaris)(89.1%),mouse(Mus musculus)(84.4%)and rabbits(Oryctolagus cuniculus)(83.7%).IHC results showed thatβ-NGF was found in the hypothalamic cells,chromophores and chromophobe cells of pituitary,ovarian granulosa cells,membranous cells,and mesenchymal cells,the endometrial glandular epithelial cells of uterus and mucosal epithelial cells of oviduct from bactrian camel.qRT-PCR results illustrated thatβ-NGF was the highest in the uterine horns,and it was extremely significantly higher than other tissues(P<0.01).The e

关 键 词：双峰驼 Β-神经生长因子 基因克隆 相对表达 

分 类 号：S814.1[农业科学—畜牧学]