精子特异性乳酸脱氢酶DNA疫苗的粘膜免疫及其效果的动物实验研究

发布时间：2018-04-11 17:47

本文选题：精子特异性乳酸脱氢酶(LDHC4) + 基因克隆　；参考：《福建医科大学》2006年硕士论文

【摘要】： 精子特异性乳酸脱氢酶通常由四个C亚基组成,因此又称为乳酸脱氢酶C4(lactate dehydrogenase C4,LDH-C4),它特异地存在于哺乳动物发育成熟的睾丸组织中,是精子能量代谢的一个关键酶,与精子的生成、代谢、获能等有密切关系。LDH-C4具有很强的免疫原性。动物实验表明,LDH-C4可在多种哺乳动物体内诱导抗LDH-C4抗体产生,后者可导致动物的生育率明显降低,且作用可逆。由于生殖道富含粘膜组织,利用LDH-C4的精子特异性及其良好的免疫原性,从粘膜途径进行DNA免疫有可能达到理想的避孕效果。本研究利用分子克隆技术,以小鼠睾丸总RNA为模板,PCR扩增小鼠精子特异性乳酸脱氢酶(mLDH-C4)编码序列,并将其插入真核表达载体pVAX1,构建真核重组表达载体pVAX1-mLDHC。利用HeLa细胞转染试验及兔网织红细胞T7快速偶联的转录/翻译系统检测pVAX1-mLDHC及pVAX1-hLDHC(本室制备好)的表达效果。采用粘膜途径免疫BALB/c小鼠(用空质粒pVAX1作对照),用Western blot与ELISA检测免疫动物血清及阴道分泌物。结果表明,所构建的DNA疫苗pVAX1-mLDHC、pVAX1-hLDHC可在雌雄两性小鼠体内及粘膜局部诱导产生特异性抗mLDH-C4或hLDH-C4抗体。LDH-C4对于雄性动物是一种自身抗原,但可能是血睾屏障的作用,雄性小鼠免疫接种后并没有产生睾丸炎。小鼠交配实验表明pVAX1-mLDHC、pVAX1-hLDHC所诱导的抗LDH-C4免疫反应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.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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