發(fā)布時(shí)間：2018-04-11 17:47

  本文選題：精子特異性乳酸脫氫酶(LDHC4) + 基因克隆�。� 參考：《福建醫(yī)科大學(xué)》2006年碩士論文

【摘要】： 精子特異性乳酸脫氫酶通常由四個(gè)C亞基組成,因此又稱為乳酸脫氫酶C4(lactate dehydrogenase C4,LDH-C4),它特異地存在于哺乳動(dòng)物發(fā)育成熟的睪丸組織中,是精子能量代謝的一個(gè)關(guān)鍵酶,與精子的生成、代謝、獲能等有密切關(guān)系。LDH-C4具有很強(qiáng)的免疫原性。動(dòng)物實(shí)驗(yàn)表明,LDH-C4可在多種哺乳動(dòng)物體內(nèi)誘導(dǎo)抗LDH-C4抗體產(chǎn)生,后者可導(dǎo)致動(dòng)物的生育率明顯降低,且作用可逆。由于生殖道富含粘膜組織,利用LDH-C4的精子特異性及其良好的免疫原性,從粘膜途徑進(jìn)行DNA免疫有可能達(dá)到理想的避孕效果。 本研究利用分子克隆技術(shù),以小鼠睪丸總RNA為模板,PCR擴(kuò)增小鼠精子特異性乳酸脫氫酶(mLDH-C4)編碼序列,并將其插入真核表達(dá)載體pVAX1,構(gòu)建真核重組表達(dá)載體pVAX1-mLDHC。利用HeLa細(xì)胞轉(zhuǎn)染試驗(yàn)及兔網(wǎng)織紅細(xì)胞T7快速偶聯(lián)的轉(zhuǎn)錄/翻譯系統(tǒng)檢測(cè)pVAX1-mLDHC及pVAX1-hLDHC(本室制備好)的表達(dá)效果。采用粘膜途徑免疫BALB/c小鼠(用空質(zhì)粒pVAX1作對(duì)照),用Western blot與ELISA檢測(cè)免疫動(dòng)物血清及陰道分泌物。結(jié)果表明,所構(gòu)建的DNA疫苗pVAX1-mLDHC、pVAX1-hLDHC可在雌雄兩性小鼠體內(nèi)及粘膜局部誘導(dǎo)產(chǎn)生特異性抗mLDH-C4或hLDH-C4抗體。LDH-C4對(duì)于雄性動(dòng)物是一種自身抗原,但可能是血睪屏障的作用,雄性小鼠免疫接種后并沒有產(chǎn)生睪丸炎。小鼠交配實(shí)驗(yàn)表明pVAX1-mLDHC、pVAX1-hLDHC所誘導(dǎo)的抗LDH-C4免疫反應(yīng)均能引起小鼠生育率下
[Abstract]:Sperm specific lactate dehydrogenase is usually composed of four subunits of C, so it is also called lactate dehydrogenase (C4(lactate dehydrogenase C4) LDH-C4. It is a key enzyme of sperm energy metabolism and spermatogenesis, which is specifically present in the mature testis of mammals.Metabolism, capacitation, etc. LDH-C4 has strong immunogenicity.Animal experiments have shown that LDH-C4 can induce the production of anti LDH-C4 antibodies in many mammals, the latter can significantly reduce the fertility rate of animals, and the effect is reversible.Because the genital tract is rich in mucosal tissue, the sperm specificity and immunogenicity of LDH-C4 can be used to immunize DNA from the mucosal pathway.In this study, the specific lactate dehydrogenase mLDH-C4 (mLDH-C4) encoding sequence of mouse spermatozoa was amplified by molecular cloning technique using total mouse testis RNA as template, and inserted into eukaryotic expression vector pVAX1 to construct eukaryotic recombinant expression vector pVAX1-mLDHC.The expression of pVAX1-mLDHC and pVAX1-hLDHCwas detected by HeLa cell transfection test and rabbit reticulocyte T7 coupled transcription / translation system.BALB/c mice were immunized with mucosal pathway (blank plasmid pVAX1 was used as control. Western blot and ELISA were used to detect serum and vaginal secretions of immunized mice.The results showed that the constructed DNA vaccine pVAX1-mLDHC-pVAX1-hLDHC could induce the production of specific anti-#en1# or hLDH-C4 antibody. LDH-C4 was an autoantigen in male animals, but it might be the function of blood-testis barrier.Male mice did not develop orchitis after immunization.Mouse mating test showed that pVAX1-mLDHCH induced anti-#en0# immunoreaction induced by pVAX1-hLDHC could induce the birth rate of mice.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392

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