本文選題：KISS1基因 + 免疫去勢��； 參考：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：吻素(KISS1)通過控制下丘腦Gn RH神經(jīng)元分泌Gn RH調(diào)控生殖軸,進而在動物繁殖功能方面起到至關(guān)重要的開關(guān)作用。KISS1基因已經(jīng)被證明能夠作為基因免疫去勢良好的靶標。前期試驗采用質(zhì)粒肌肉注射的方式接種公羔取得了較為理想的去勢效果,但是質(zhì)粒的制備存在繁瑣和價格較高等問題。因此,需要構(gòu)建一種制備簡單、實用性強的新型KISS1基因疫苗。本研究將前期構(gòu)建的p VAX-KISS1-S-asd真核表達質(zhì)粒通過電轉(zhuǎn)化的方式轉(zhuǎn)入減毒豬霍亂沙門氏菌C500中,經(jīng)測序和酶切鑒定挑選陽性克隆,命名為C500/p KS-asd;將重組菌C500/p KS-asd在無菌的條件下連續(xù)接種50次,提取質(zhì)粒和細菌基因組DNA,通過質(zhì)粒雙酶切和沙門氏菌屬中保守Inv A基因序列的PCR擴增鑒定重組真核表達質(zhì)粒和重組菌株的傳代穩(wěn)定性。用C500/p KS-asd和C500/p VAX-asd重組菌通過滴鼻方式分3次免疫10只10周齡的公羔,每次間隔4周,劑量為2×1010CFU/只,每隔兩周采集血樣和測量陰囊周長;初次免疫后14周屠宰試驗羊,采集組織樣和血樣。用ELISA的方法檢測血清中的抗體和生殖激素;用HE染色的方法制作睪丸切片;用實時熒光定量PCR的方法分析對照組與試驗組公羊中下丘腦-垂體-睪丸軸上相關(guān)生殖基因相對表達量的差異。結(jié)果表明,重組菌C500/p KS-asd和重組真核表達質(zhì)粒均具有良好的傳代穩(wěn)定性。試驗組從初次免疫后第4周至14周檢測到抗體水平逐次增高。試驗組睪酮激素濃度呈降低趨勢,在初次免疫后第8周至12周顯著低于對照組(P0.05)。陰囊周長從初次免疫后第2周至14周也均低于對照組(P0.05)。初次免疫后第12-14周試驗組公羊性行為表現(xiàn)(嗅聞、爬跨和頂撞頻率)明顯低于對照組(P0.05)。初次免疫后第14周屠宰時試驗組公羊的睪丸大小(長度、寬度和體積)和重量均顯著低于對照組睪丸(P0.05);同時睪丸HE組織學(xué)檢測顯示試驗組公羊睪丸曲細精管管腔內(nèi)(生精小管ST)的精原細胞和精子產(chǎn)生量明顯少于對照組,各級生殖細胞之間較為模糊,排列松散。試驗組下丘腦中KISS1 m RNA和GPR54 m RNA相對表達量顯著高于對照組(P0.05),試驗組垂體中GPR54 m RNA、Gn RHR m RNA和LHβm RNA相對表達量顯著低于對照組(P0.05),試驗組睪丸中AR m RNA相對表達量顯著低于對照組(P0.05)。本研究成功構(gòu)建了非抗性篩選C500/p VAX-KISS1-S-asd重組菌,菌株和質(zhì)粒傳代都很穩(wěn)定;公羔通過滴鼻方式接種C500/p KS-asd重組菌后睪丸發(fā)育和精子發(fā)生受到抑制,功能去勢效果明顯。
[Abstract]:KISS1) regulates the reproductive axis by controlling the secretion of GnRH from GnRH neurons in the hypothalamus and thus plays a crucial switching role in animal reproductive function. KISS1 gene has been proved to be a good target for gene immune castration. In the previous experiment, the male lambs were inoculated with plasmids intramuscularly, but the preparation of plasmids was complicated and expensive. Therefore, it is necessary to construct a new KISS1 gene vaccine with simple preparation and strong practicability. In this study, the previously constructed eukaryotic expression plasmid of p VAX-KISS1-S-asd was transformed into attenuated porcine Salmonella cholerae C500 by electrotransformation, and the positive clones were selected by sequencing and enzyme digestion. It's called C500 / p KS-asd.The recombinant strain C500 / p KS-asd is inoculated 50 times continuously under aseptic conditions. The plasmid and bacterial genomic DNAs were extracted, and the stability of the recombinant eukaryotic expression plasmid and the recombinant strain was identified by restriction endonuclease digestion and PCR amplification of the conserved Inv A gene sequence in Salmonella. Ten 10-week-old male lambs were immunized with C500 / p KS-asd and C500 / p VAX-asd by nasal drip for 4 weeks at a dose of 2 脳 1010CFU/. Blood samples were collected and scrotal circumference was measured every two weeks. The sheep were slaughtered 14 weeks after the first immunization. Tissue and blood samples were collected. The antibody and reproductive hormone in serum were detected by ELISA, and the testicular sections were made by HE staining. The difference of relative expression of related reproductive genes on hypothalamus-pituitary-testicular axis between the control group and the experimental group was analyzed by real-time fluorescence quantitative PCR. The results showed that both the recombinant C500 / p KS-asd and the recombinant eukaryotic expression plasmid had good passage stability. The antibody level in the test group increased successively from the 4th week to the 14th week after the first immunization. The concentration of testosterone in the experimental group showed a decreasing trend and was significantly lower than that in the control group from the 8th week to the 12th week after the first immunization. The circumference of scrotum was also lower than that of control group from the 2nd week to 14th week after primary immunization. At 12-14 weeks after primary immunization, the sexual behavior (smelling, crawling and collision frequency) of the male sheep in the experimental group was significantly lower than that in the control group (P 0.05). Testicular size (length) of the male sheep of the experimental group at slaughter at the 14th week after primary immunization, The width, volume and weight of testis were significantly lower than that of the control group (P 0.05), and the testis HE histological examination showed that the spermatogonia and spermatozoa production in testicular tubule (ST1) of male sheep were significantly lower than that of control group. The germ cells at all levels are fuzzy and loosely arranged. The relative expression of KISS1 m RNA and GPR54 m RNA in the hypothalamus of the experimental group was significantly higher than that in the control group (P 0.05), the relative expression of GPR54 m RNA-G n RHR m RNA and LH 尾 m RNA in the pituitary gland of the test group was significantly lower than that in the control group (P 0.05), and the relative expression of AR m RNA in the testis of the experimental group was significantly lower than that in the control group (P 0.05). In this study, the non-resistant screening of C500 / p VAX-KISS1-S-asd recombinant bacteria was successfully constructed, the strain and plasmid passage were stable, and the testis development and spermatogenesis were inhibited after male lambs were inoculated with C500 / p KS-asd recombinant bacteria by nasal drip, and the functional ovariectomized effect was obvious.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.4

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