本文選題：抑制素 + 真核表達(dá)載體��； 參考：《石河子大學(xué)》2015年碩士論文

【摘要】：抑制素(inhibin,INH)又稱(chēng)卵泡抑制素或性腺抑制素,是一種主要由卵巢顆粒細(xì)胞和睪丸支持細(xì)胞分泌產(chǎn)生的糖蛋白質(zhì)激素,由一個(gè)α亞基和一個(gè)β亞基通過(guò)二硫鍵構(gòu)成,其中抑制素α亞基(INHα)是抑制素發(fā)揮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)的釋放,對(duì)雌性動(dòng)物主要表現(xiàn)為調(diào)節(jié)卵泡的生成,增加成熟的優(yōu)勢(shì)卵泡數(shù)量;對(duì)雄性動(dòng)物主要表現(xiàn)為促進(jìn)精子的發(fā)生,提高睪丸的生精能力;還對(duì)雌雄動(dòng)物共同表現(xiàn)為促使性成熟提前。因此,可通過(guò)調(diào)節(jié)抑制素α亞基基因在機(jī)體內(nèi)的表達(dá)量,降低體內(nèi)抑制素水平,促進(jìn)卵泡發(fā)育和精子的生成,最終提高動(dòng)物的繁殖力。目前主要通過(guò)兩種途徑降低動(dòng)物體內(nèi)抑制素的水平,其一是通過(guò)對(duì)動(dòng)物進(jìn)行主動(dòng)或被動(dòng)免疫抑制素原來(lái)使得機(jī)體內(nèi)抑制素抗體含量增加,中和動(dòng)物體內(nèi)所分泌的抑制素,從而降低動(dòng)物機(jī)體內(nèi)的抑制素水平;其二是通過(guò)RNA干擾技術(shù)下調(diào)抑制素α亞基基因m RNA水平上的表達(dá),最終致使動(dòng)物機(jī)體內(nèi)所分泌抑制素的量相對(duì)減少,降低在動(dòng)物體內(nèi)的抑制素水平。本研究運(yùn)用了生物信息學(xué)技術(shù)、基因克隆技術(shù)、蛋白質(zhì)分析技術(shù)、基因免疫技術(shù)等以新疆塔城地區(qū)也木勒白羊卵泡抑制素α亞基作為選擇基因,對(duì)目的基因主要進(jìn)行以下幾方面的研究試驗(yàn):1、也木勒白羊卵泡抑制素α亞基基因的克隆及序列分析為了克隆和序列分析也木勒白羊抑制素α亞基基因,根據(jù)Gen Bank檢索到的INHα亞基基因序列(Gen Bank號(hào):XM_004004955.1)設(shè)計(jì)一對(duì)特異性引物,本試驗(yàn)采集發(fā)情期也木勒白羊卵巢組織為研究對(duì)象,從中快速抽提總RNA作為模板。利用基因克隆技術(shù)和現(xiàn)代生物技術(shù)對(duì)也木勒白羊INHαc DNA進(jìn)行克隆和測(cè)序及序列分析。測(cè)序及分析結(jié)果:也木勒白羊抑制素基因全長(zhǎng)為1109bp,其中只含有1083bp組成開(kāi)放閱讀框(ORF),其起始密碼子為ATG,終止密碼子為T(mén)AA,含完整的抑制素α亞基亞基序列編碼區(qū),共編碼360個(gè)氨基酸殘基。將所得序列提交到Gen Bank,獲得登錄號(hào):KP-113678。2、也木勒白羊卵泡抑制素α亞基基因真核表達(dá)載體的構(gòu)建對(duì)也木勒白羊抑制素α亞基基因進(jìn)行克隆和序列分析的基礎(chǔ)上,采用定向克隆技術(shù),構(gòu)建也木勒白羊卵泡抑制素α亞基基因與綠色熒光蛋白基因融合表達(dá)載體p EGFP-INHα,并對(duì)所構(gòu)建的真核表達(dá)載體p EGFP-INHα,運(yùn)用菌液PCR、雙酶切、測(cè)序、BHK-21細(xì)胞瞬轉(zhuǎn)及蛋白表達(dá)等試驗(yàn)進(jìn)行鑒定。結(jié)果:本次試驗(yàn)成功構(gòu)建了含也木勒白羊卵泡抑制素α亞基基因的綠色熒光真核表達(dá)載體(p EGFP-INHα),并在BHK-21細(xì)胞中獲得了分泌型表達(dá),轉(zhuǎn)染率高達(dá)70%以上。檢測(cè)到的該基因蛋白表達(dá)分子量約為40KD,該載體的構(gòu)建為抑制素基因疫苗的研制奠定了一定基礎(chǔ)。3、也木勒白羊卵泡抑制素α亞基基因疫苗對(duì)家兔生殖激素的影響為了初步研究構(gòu)建的也木勒白羊卵泡抑制素α亞基綠色熒光真核表達(dá)載體(p EGFP-INHα)的免疫原性,在本實(shí)驗(yàn)中根據(jù)基因免疫技術(shù),以成功制備的抑制素重組質(zhì)粒p EGFP-INHα為免疫原,對(duì)30只家兔進(jìn)行基因免疫試驗(yàn),應(yīng)用酶聯(lián)免疫法(ELISA)檢測(cè)抗抑制素抗體,放射免疫法(RIA)測(cè)定處理后不同時(shí)期血清中部分生殖激素水平。結(jié)果:重組質(zhì)粒p EGFP-INHα免疫家兔后,各實(shí)驗(yàn)組抗體滴度均顯著高于對(duì)照組(P0.05),且在第2次加強(qiáng)免疫后抗體水平明顯上升。首次免疫后,促卵泡素(FSH)和雌二醇(E2)平均含量均高于對(duì)照組,且在第2次加強(qiáng)免疫階段差異顯著(P0.05),但在兩次免疫前后促黃體素(LH)均無(wú)明顯變化。這些結(jié)果表明:抑制素基因免疫家兔可降低抑制素的含量,促進(jìn)FSH和E2分泌,進(jìn)而影響動(dòng)物卵泡發(fā)育和誘導(dǎo)動(dòng)物多產(chǎn)。4、也木勒白羊卵泡抑制素α亞基基因靶向Si RNA真核表達(dá)載體的構(gòu)建為了構(gòu)建也木勒白羊抑制素α亞基基因靶向Si RNA真核表達(dá)載體,根據(jù)本課題上傳至Gen Bank的INHα基因序列(Gen Bank號(hào):KP-113678.1)設(shè)計(jì)3個(gè)不同位點(diǎn)的干擾片段,利用RNAi技術(shù)和實(shí)驗(yàn)室常規(guī)方法將3個(gè)干擾片段同時(shí)連接到同一個(gè)小RNA專(zhuān)用真核表達(dá)載體中,并對(duì)其進(jìn)行相關(guān)檢測(cè)、鑒定及干擾效率評(píng)價(jià)。結(jié)果:干擾表達(dá)質(zhì)粒能在綿羊顆粒細(xì)胞中成功表達(dá),且轉(zhuǎn)染率在60%左右,通過(guò)Q-PCR和蛋白表達(dá)試驗(yàn),發(fā)現(xiàn)試驗(yàn)組與對(duì)照組相比,試驗(yàn)組沉默效率達(dá)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%.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S826

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