人精子特异性乳酸脱氢酶的原核表达、DNA疫苗的构建及其基因免疫效果的初步鉴定

发布时间：2018-12-25 18:27

【摘要】：精子特异性乳酸脱氢酶,即乳酸脱氢酶C4(lactate dehydrogenaseC4,LDH-C4),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。LDH-C4的精子特异性及其良好的免疫原性使其成为一个很好的避孕疫苗候选对象。本研究利用分子克隆技术,以人睾丸λTripEx cDNA文库为模板,PCR扩增人精子特异性乳酸脱氢酶(hLDH-C4)编码序列,并将其插入原核表达载体pET-28a(+),构建原核重组表达载体pET-28a(+)—hLDHC4,在E.coli BL21(DE3 plysS+)中诱导hLDH-C4的His-融合蛋白的表达。通过12%SDS聚丙烯酰胺凝胶电泳、蛋白免疫印迹、酶活性测定和酶谱分析鉴定表达产物。结果表明,重组hLDH-C4在E.coli中获得较高表达,并具有较高的乳酸脱氢酶活性。用Ni螯和层析柱进行分离纯化,得到了纯度较高的重组蛋白,为特异性抗LDH-C4抗体的检测打下了基础。以原核表达载体pET-28a(+)-hLDHC4为模板,PCR扩增人精子特异性乳酸脱氢酶(hLDH-C4)编码序列,插入真核表达载体pVAX1,构建DNA疫苗pVAX1-hLDHC4。利用兔网织红细胞T7快速偶联的转录/翻译系统检测其表达效果。结果表明,重组载体在该系统中能指导hLDH-C4合成,后者表现出较
[Abstract]:Spermatozoa specific lactate dehydrogenase C4 (lactate dehydrogenaseC4,LDH-C4), a key enzyme in sperm energy metabolism, is present in mammalian testis, and is associated with spermatogenesis and metabolism. LDH-C4 has strong immunogenicity. Animal experiments have shown that LDH-C4 can induce the production of anti LDH-C4 antibodies in a variety of mammals, which can significantly reduce the fertility rate of animals. The sperm specificity and immunogenicity of LDH-C4 make it a good candidate for contraceptive vaccine. In this study, using human testis 位 TripEx cDNA library as template, the coding sequence of human spermatozoa specific lactate dehydrogenase (hLDH-C4) was amplified by molecular cloning and inserted into prokaryotic expression vector pET-28a (),. A prokaryotic recombinant expression vector pET-28a ()-hLDHC4, was constructed to induce the expression of hLDH-C4 His- fusion protein in E.coli BL21 (DE3 plysS). The expressed products were identified by 12%SDS polyacrylamide gel electrophoresis, Western blot, enzyme activity and zymogram analysis. The results showed that the recombinant hLDH-C4 was highly expressed in E.coli and had high lactate dehydrogenase activity. The recombinant protein with high purity was obtained by Ni chelating and chromatography column, which laid a foundation for the detection of specific anti LDH-C4 antibody. Using prokaryotic expression vector pET-28a ()-hLDHC4 as template, the coding sequence of human spermatozoa specific lactate dehydrogenase (hLDH-C4) was amplified by PCR and inserted into eukaryotic expression vector pVAX1, to construct DNA vaccine pVAX1-hLDHC4.. The expression of rabbit reticulocyte T 7 was detected by a fast coupled transcription / translation system. The results showed that the recombinant vector could guide the synthesis of hLDH-C4 in the system, and the latter showed a better performance.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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