【摘要】：精子特異性乳酸脫氫酶,即乳酸脫氫酶C4(lactate dehydrogenaseC4,LDH-C4),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。LDH-C4的精子特異性及其良好的免疫原性使其成為一個(gè)很好的避孕疫苗候選對(duì)象。 本研究利用分子克隆技術(shù),以人睪丸λTripEx cDNA文庫(kù)為模板,PCR擴(kuò)增人精子特異性乳酸脫氫酶(hLDH-C4)編碼序列,并將其插入原核表達(dá)載體pET-28a(+),構(gòu)建原核重組表達(dá)載體pET-28a(+)—hLDHC4,在E.coli BL21(DE3 plysS+)中誘導(dǎo)hLDH-C4的His-融合蛋白的表達(dá)。通過(guò)12%SDS聚丙烯酰胺凝膠電泳、蛋白免疫印跡、酶活性測(cè)定和酶譜分析鑒定表達(dá)產(chǎn)物。結(jié)果表明,重組hLDH-C4在E.coli中獲得較高表達(dá),并具有較高的乳酸脫氫酶活性。用Ni螯和層析柱進(jìn)行分離純化,得到了純度較高的重組蛋白,為特異性抗LDH-C4抗體的檢測(cè)打下了基礎(chǔ)。 以原核表達(dá)載體pET-28a(+)-hLDHC4為模板,PCR擴(kuò)增人精子特異性乳酸脫氫酶(hLDH-C4)編碼序列,插入真核表達(dá)載體pVAX1,構(gòu)建DNA疫苗pVAX1-hLDHC4。利用兔網(wǎng)織紅細(xì)胞T7快速偶聯(lián)的轉(zhuǎn)錄/翻譯系統(tǒng)檢測(cè)其表達(dá)效果。結(jié)果表明,重組載體在該系統(tǒng)中能指導(dǎo)hLDH-C4合成,后者表現(xiàn)出較
[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.
【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】：2005
【分類號(hào)】：R392

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