也木勒白羊卵泡抑制素α亚基DNA疫苗免疫原性及RNA干扰效果初步研究

发布时间：2018-05-31 06:59

本文选题：抑制素 + 真核表达载体　；参考：《石河子大学》2015年硕士论文

【摘要】：抑制素(inhibin,INH)又称卵泡抑制素或性腺抑制素,是一种主要由卵巢颗粒细胞和睾丸支持细胞分泌产生的糖蛋白质激素,由一个α亚基和一个β亚基通过二硫键构成,其中抑制素α亚基(INHα)是抑制素发挥生理功能必不可少的。抑制素主要生理功能是抑制促卵泡素(FSH)的释放,对雌性动物主要表现为调节卵泡的生成,增加成熟的优势卵泡数量;对雄性动物主要表现为促进精子的发生,提高睾丸的生精能力;还对雌雄动物共同表现为促使性成熟提前。因此,可通过调节抑制素α亚基基因在机体内的表达量,降低体内抑制素水平,促进卵泡发育和精子的生成,最终提高动物的繁殖力。目前主要通过两种途径降低动物体内抑制素的水平,其一是通过对动物进行主动或被动免疫抑制素原来使得机体内抑制素抗体含量增加,中和动物体内所分泌的抑制素,从而降低动物机体内的抑制素水平;其二是通过RNA干扰技术下调抑制素α亚基基因m RNA水平上的表达,最终致使动物机体内所分泌抑制素的量相对减少,降低在动物体内的抑制素水平。本研究运用了生物信息学技术、基因克隆技术、蛋白质分析技术、基因免疫技术等以新疆塔城地区也木勒白羊卵泡抑制素α亚基作为选择基因,对目的基因主要进行以下几方面的研究试验:1、也木勒白羊卵泡抑制素α亚基基因的克隆及序列分析为了克隆和序列分析也木勒白羊抑制素α亚基基因,根据Gen Bank检索到的INHα亚基基因序列(Gen Bank号:XM_004004955.1)设计一对特异性引物,本试验采集发情期也木勒白羊卵巢组织为研究对象,从中快速抽提总RNA作为模板。利用基因克隆技术和现代生物技术对也木勒白羊INHαc DNA进行克隆和测序及序列分析。测序及分析结果:也木勒白羊抑制素基因全长为1109bp,其中只含有1083bp组成开放阅读框(ORF),其起始密码子为ATG,终止密码子为TAA,含完整的抑制素α亚基亚基序列编码区,共编码360个氨基酸残基。将所得序列提交到Gen Bank,获得登录号:KP-113678。2、也木勒白羊卵泡抑制素α亚基基因真核表达载体的构建对也木勒白羊抑制素α亚基基因进行克隆和序列分析的基础上,采用定向克隆技术,构建也木勒白羊卵泡抑制素α亚基基因与绿色荧光蛋白基因融合表达载体p EGFP-INHα,并对所构建的真核表达载体p EGFP-INHα,运用菌液PCR、双酶切、测序、BHK-21细胞瞬转及蛋白表达等试验进行鉴定。结果:本次试验成功构建了含也木勒白羊卵泡抑制素α亚基基因的绿色荧光真核表达载体(p EGFP-INHα),并在BHK-21细胞中获得了分泌型表达,转染率高达70%以上。检测到的该基因蛋白表达分子量约为40KD,该载体的构建为抑制素基因疫苗的研制奠定了一定基础。3、也木勒白羊卵泡抑制素α亚基基因疫苗对家兔生殖激素的影响为了初步研究构建的也木勒白羊卵泡抑制素α亚基绿色荧光真核表达载体(p EGFP-INHα)的免疫原性,在本实验中根据基因免疫技术,以成功制备的抑制素重组质粒p EGFP-INHα为免疫原,对30只家兔进行基因免疫试验,应用酶联免疫法(ELISA)检测抗抑制素抗体,放射免疫法(RIA)测定处理后不同时期血清中部分生殖激素水平。结果:重组质粒p EGFP-INHα免疫家兔后,各实验组抗体滴度均显著高于对照组(P0.05),且在第2次加强免疫后抗体水平明显上升。首次免疫后,促卵泡素(FSH)和雌二醇(E2)平均含量均高于对照组,且在第2次加强免疫阶段差异显著(P0.05),但在两次免疫前后促黄体素(LH)均无明显变化。这些结果表明:抑制素基因免疫家兔可降低抑制素的含量,促进FSH和E2分泌,进而影响动物卵泡发育和诱导动物多产。4、也木勒白羊卵泡抑制素α亚基基因靶向Si RNA真核表达载体的构建为了构建也木勒白羊抑制素α亚基基因靶向Si RNA真核表达载体,根据本课题上传至Gen Bank的INHα基因序列(Gen Bank号:KP-113678.1)设计3个不同位点的干扰片段,利用RNAi技术和实验室常规方法将3个干扰片段同时连接到同一个小RNA专用真核表达载体中,并对其进行相关检测、鉴定及干扰效率评价。结果:干扰表达质粒能在绵羊颗粒细胞中成功表达,且转染率在60%左右,通过Q-PCR和蛋白表达试验,发现试验组与对照组相比,试验组沉默效率达83%。
[Abstract]:Inhibin (INH), also known as follicle inhibin or gonadin, is a glycoprotein hormone produced mainly by ovarian granulosa cells and testis supporting cells. It is made up of an alpha subunit and a beta subunit through the two sulfur bond, in which the inhibin alpha subunit (INH alpha) is essential to the function of inhibin. The physiological function is to inhibit the release of follicle stimulating hormone (FSH) and to regulate the formation of follicles and increase the number of mature follicles in the female animals; the male animals are mainly shown to promote the occurrence of sperm and improve the spermatogenesis of the testis. The expression of hormone alpha subunit gene in the body, reducing the level of inhibin in the body, promoting the development of follicle and the production of sperm, and ultimately improving the fecundity of animals. At present, the level of inhibin in animals is reduced mainly through two ways. One is to make inhibin in vivo by active or passive immune inhibin. The antibody content is increased and the inhibin secreted in the animal's body reduces the inhibin level in the animal body, and the second is to decrease the level of inhibin secreted by the inhibin alpha subunit gene m RNA through the RNA interference technique, and reduce the level of inhibin in the animal body. Using bioinformatics, gene cloning, protein analysis, and gene immunization, the subunit of follicular inhibin alpha subunit in Tacheng, Xinjiang, was used as the selection gene, and the main target genes were studied in the following aspects: 1, the cloning and sequence of the follicular inhibin alpha subunit gene of Muller white goat. In order to clone and analyze the clonal alpha subunit gene of the Mulle white sheep inhibin alpha, a pair of specific primers was designed based on the INH alpha subunit gene sequence (Gen Bank No. Bank: XM_004004955.1) retrieved by Gen Bank. This experiment collected the oestrus of Mulle ovaries in the oestrus period as the research object, and quickly extracted the total RNA as a template. The cloning and sequencing and sequence analysis of INH alpha C DNA of Mulle white sheep were sequenced and sequenced. The results of sequencing and analysis showed that the total length of the Mulle white sheep inhibin gene was 1109bp, which contained only the 1083bp composition open reading frame (ORF), the starting codon was ATG, the terminating codon was TAA, and the complete inhibin alpha subunit sequence encoding was encoded. A total of 360 amino acid residues were encoded in the region. The sequence was submitted to Gen Bank to obtain the login number: KP-113678.2, and the construction of the eukaryotic expression vector of the follicular inhibin alpha subunit gene was constructed on the basis of cloning and sequence analysis of the mullera white sheep inhibin alpha subunit gene. The expression vector p EGFP-INH alpha was fused with the expression vector of the inhibin alpha subunit and the green fluorescent protein gene, and the constructed eukaryotic expression vector p EGFP-INH a was identified by the test of bacterial liquid PCR, double enzyme digestion, sequencing, BHK-21 cell transient and protein expression. The green fluorescent eukaryotic expression vector (P EGFP-INH alpha) was expressed in BHK-21 cells, and the transfection rate was up to 70%. The molecular weight of the gene protein was about 40KD. The construction of the carrier laid a foundation.3 for the development of the inhibin gene vaccine, and the gene plague of the follicular inhibin alpha subunit of the Muller white goat. In order to study the immunogenicity of P EGFP-INH alpha, a green fluorescent eukaryotic expression vector (P EGFP-INH alpha) of the follicular inhibin alpha subunit of Muller white sheep, the effect of the vaccine on the reproductive hormones of the rabbit was preliminarily studied. In this experiment, the recombinant plasmid P EGFP-INH a, which was successfully prepared by the gene immunization, was used as immunogen to 30 rabbits to be immunized. The anti inhibin antibody was detected by enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) was used to determine the level of some reproductive hormones in the serum of different periods after treatment. Results: after the recombinant plasmid P EGFP-INH alpha was immunized with rabbits, the antibody titers of all the experimental groups were significantly higher than those of the control group (P0.05), and the antibody level was obviously increased after second times of immunization. After subimmunization, the average content of follicle stimulating hormone (FSH) and estradiol (E2) was higher than that of the control group, and the difference was significant (P0.05) at the second stage of immunization, but there was no significant change in the luteinizing hormone (LH) before and after the two immunization. These results showed that the inhibin gene immunological rabbit could reduce the content of inhibin, promote the secretion of FSH and E2, and then affect the activity. The development of follicular follicles and the induction of Prolificacy of.4, and the construction of the eukaryotic expression vector targeting Si RNA by the follicle inhibin alpha subunit gene of Muller white sheep in order to construct the eukaryotic expression vector of Si RNA, which is also designed to target the INH a gene sequence of Gen Bank (Gen Bank: KP-113678.1) according to this subject. The interference fragment, using RNAi and laboratory routine methods, connects 3 interference fragments to the same small RNA eukaryotic expression vector, and carries out correlation detection, identification and interference efficiency evaluation. Results: interference expression plasmids can be expressed in sheep granulosa cells, and the transfection rate is about 60%, through Q-PCR and eggs. White expression test showed that compared with the control group, the experimental group had a silencing efficiency of 83%.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S826

【参考文献】