【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物發(fā)育成熟的睪丸組織中,是精子能量代謝的一個關(guān)鍵酶,與精子的生成、代謝、獲能等有密切關(guān)系。LDH-C4具有很強的免疫原性。動物實驗表明,LDH-C4可在多種哺乳動物體內(nèi)誘導(dǎo)抗LDH-C4抗體產(chǎn)生,后者可導(dǎo)致動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很好的避孕疫苗候選對象。 本研究利用分子克隆技術(shù),以人睪丸λTripEx cDNA文庫為模板,PCR擴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載體pET-28a(+),構(gòu)建原核重組表達載體pET-28a(+)—hLDHC4,在E.coli BL21(DE3 plysS+)中誘導(dǎo)hLDH-C4的His-融合蛋白的表達。通過12%SDS聚丙烯酰胺凝膠電泳、蛋白免疫印跡、酶活性測定和酶譜分析鑒定表達產(chǎn)物。結(jié)果表明,重組hLDH-C4在E.coli中獲得較高表達,并具有較高的乳酸脫氫酶活性。用Ni螯和層析柱進行分離純化,得到了純度較高的重組蛋白,為特異性抗LDH-C4抗體的檢測打下了基礎(chǔ)。 以原核表達載體pET-28a(+)-hLDHC4為模板,PCR擴增人精子特異性乳酸脫氫酶(hLDH-C4)編碼序列,插入真核表達載體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)檢測其表達效果。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)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R392

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