非抗性筛选KISS1基因疫苗构建及其免疫效果的研究

发布时间：2018-05-21 02:16

本文选题：KISS1基因 + 免疫去势　；参考：《华中农业大学》2017年硕士论文

【摘要】：吻素(KISS1)通过控制下丘脑Gn RH神经元分泌Gn RH调控生殖轴,进而在动物繁殖功能方面起到至关重要的开关作用。KISS1基因已经被证明能够作为基因免疫去势良好的靶标。前期试验采用质粒肌肉注射的方式接种公羔取得了较为理想的去势效果,但是质粒的制备存在繁琐和价格较高等问题。因此,需要构建一种制备简单、实用性强的新型KISS1基因疫苗。本研究将前期构建的p VAX-KISS1-S-asd真核表达质粒通过电转化的方式转入减毒猪霍乱沙门氏菌C500中,经测序和酶切鉴定挑选阳性克隆,命名为C500/p KS-asd;将重组菌C500/p KS-asd在无菌的条件下连续接种50次,提取质粒和细菌基因组DNA,通过质粒双酶切和沙门氏菌属中保守Inv A基因序列的PCR扩增鉴定重组真核表达质粒和重组菌株的传代稳定性。用C500/p KS-asd和C500/p VAX-asd重组菌通过滴鼻方式分3次免疫10只10周龄的公羔,每次间隔4周,剂量为2×1010CFU/只,每隔两周采集血样和测量阴囊周长;初次免疫后14周屠宰试验羊,采集组织样和血样。用ELISA的方法检测血清中的抗体和生殖激素;用HE染色的方法制作睾丸切片;用实时荧光定量PCR的方法分析对照组与试验组公羊中下丘脑-垂体-睾丸轴上相关生殖基因相对表达量的差异。结果表明,重组菌C500/p KS-asd和重组真核表达质粒均具有良好的传代稳定性。试验组从初次免疫后第4周至14周检测到抗体水平逐次增高。试验组睾酮激素浓度呈降低趋势,在初次免疫后第8周至12周显著低于对照组(P0.05)。阴囊周长从初次免疫后第2周至14周也均低于对照组(P0.05)。初次免疫后第12-14周试验组公羊性行为表现(嗅闻、爬跨和顶撞频率)明显低于对照组(P0.05)。初次免疫后第14周屠宰时试验组公羊的睾丸大小(长度、宽度和体积)和重量均显著低于对照组睾丸(P0.05);同时睾丸HE组织学检测显示试验组公羊睾丸曲细精管管腔内(生精小管ST)的精原细胞和精子产生量明显少于对照组,各级生殖细胞之间较为模糊,排列松散。试验组下丘脑中KISS1 m RNA和GPR54 m RNA相对表达量显著高于对照组(P0.05),试验组垂体中GPR54 m RNA、Gn RHR m RNA和LHβm RNA相对表达量显著低于对照组(P0.05),试验组睾丸中AR m RNA相对表达量显著低于对照组(P0.05)。本研究成功构建了非抗性筛选C500/p VAX-KISS1-S-asd重组菌,菌株和质粒传代都很稳定;公羔通过滴鼻方式接种C500/p KS-asd重组菌后睾丸发育和精子发生受到抑制,功能去势效果明显。
[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.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.4

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