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Analysis of Trichomonas Vaginalis and Mouse LAG1 Gene Promoter and Expression of an Actin cDNA Fragment from Trichomonas Vaginalis

Author: LiuJuLi
Tutor: FuYuCai
School: Shantou University
Course: Genetics
Keywords: LAG1 gene promoter actin recombinant protein expression antiserum
CLC: Q78
Type: Master's thesis
Year: 2007
Downloads: 38
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Objective: (1) To clone and analyze the Trichomonas vaginalis(Tv) LAG1 and mouse LASS1 gene promoter for investigating how LAG1(LASS1) is transcriptionally regulated in the progress of cell senescence. (2) To clone and express an actin cDNA fragment, and to prepare the antiserum for analyzing the function of actin proteins information and maintaining the cystoskeleton in Tv.Materials and Methods: (1) The region supposed to contain the promoter of LAG1(LASS1) gene was amplified by PCR, and PCR products were cloned to pGEM-T-Easy vector, and subcloned to the luciferase reporter vector pGL3-Basic and the enhanced green fluorescent reporter vector pEGFP-1. EC109 cells were transfected by these expression vectors. The luciferase activity was measured and the expression of green fluorescent protein was observed after 48h. (2) A fragment of actin gene was amplified by a pair of primer designed according to an actin cDNA sequence from Genbank, and the PCR products were cloned to pGEM- T -Easy vector, and subcloned to the expression vector pET-41. E.coli BL21 cells were transformed by the recombinant plasmid. The expression of recombinant protein was induced by IPTG. The recombinant protein was purified with GSTrap affinity columns. A guinea pig was injected with the recombinant protein for the preparation of antiserum. SDS- PAGE and Western blot analysis were performed to detect the recombinant protein with the antiserum. Results: (1) As for Tv, the constructed six luciferase expression vectors and two green fluorescent protein expression vectors, the result shows that the luciferase activities of pGL3-455(-455bp~+55bp)、pGL3-417(-417bp~+55bp)、pGL3-280(-280bp~+55bp)、pGL3-202(-202bp~+55bp) and pGL3-81 (-81bp~+55bp) were comparable, and were significantly higher than that of pGL3-47(-47~+55). And there was no evident difference between the activity of pGL3-47 and pGL3-Basic. The fluorescent protein expression of pEGFP-81(-81bp~+55bp) was very high, but those of pEGFP-47(-47~+55) and pEGFP-1 were almost scarcity. (2) As for the mouse, three luciferase expression vectors: pGL3-501(-501bp~+106bp)、pGL3-181(-181bp~+106bp) and pGL3-26(+26bp~+106bp), and three green fluorescent protein expression vectors: pEGFP-501、pEGFP-181 and pEGFP-26 were constructed. The luciferase activities of pGL3-501 and pGL3-181 were comparable, and were significantly higher than that of pGL3-26. pGL3-26 showed very little luciferase activity as pGL3-Basic did. The fluorescent protein expression of pEGFP-501 and pEGFP-181 was very high, but those of pEGFP-26 and pEGFP-1 were almost scarcity. (3) The cDNA fragment of 500bp of Tv actin gene was cloned, and the recombinant expression plasmid was constructed. The recombinant protein (about 47kDa) was expressed and purified. The antiserum reacted with the recombinant protein and detected a 41kDa in the extracts of Tv.Conclusion: (1)At the 5’ flank of Tv LAG1 gene, the region from -81bp to -47bp contains an essential promoter sequence for the gene transcription. (2)At the 5’ flank of mouse LASS1 gene, the region from -181bp to +26bp includes an essential promoter sequence for the gene transcription. The region contains two SP1 elements, which are necessary for the transcription of mouse LASS1 gene. (3) The recombinant actin protein of Tv was expressed well. The antiserum will be used to analyze the function of actin protein in formation and maintaining the cystoskeleton in Tv.

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