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Construction and Expression of Recombinant Adenovirus Carrying Human MIP-1α Gene and Preliminary Study on Its Chemotactic Functions

Author: LinChangLe
Tutor: ZengYaoYing
School: Jinan University
Course: Immunology
Keywords: MIP-1α recombined adenovirus HEK-293A chemotactic effect
CLC: R392
Type: Master's thesis
Year: 2007
Downloads: 54
Quote: 0
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Abstract


Objective:To construct the recombinant adenovirus carring the human MIP-1α(macrophageinfla-mmatory protein 1α) and study the chemotactic effect of MIP-1αon the CD3+、CD4+、CD8+ lymphocyte and monocyte, establish the foundation for the further research on thefunction and action mechanism of MIP-1αin the immune system.Methods:Obtain the MIP-1αgene by PCR with pReceiver-M01-mip as the template, and construct therecombinant plasmid pAdTrack-mip by double digestion and ligation. The pAdTrack-mip waslineared by Pme I and co-transducted in BJ5183 with pAdeasy to obtain the recombinantplasmid pAd-mip. Transfected pAd-mip in the HEK-293A cell after lineared with Pac I andcollected the recombinant adenovirus. Virus titre was detected by TCID50. Western blot and FlexSet system were used to identify the MIP-1αprotein in the HEK-293A cell and the supernatantrespectively. Build up chemotactic model with Transwell and flow cytometre absolute countingmethod, and evaluate the chemotactic effect of MIP-1αon CD3+、CD4+、CD8+ lymphocyte andmonocyte. Results:Constructed the recombinant adenovirus Ad-mip, confirm that MIP-1αprotein can be expressin the HEK-293A cell and secreted in the supernatant. After cultured with supernatant ofHEK-293A cell infected by Ad-mip 5 h, CD3+、CD4+、and monocyte become chemotacticobviously. And the monocyte had the highest chemotactic index (5.55±0.12). CD4+lymphocyte’s chemotactic index is higher than CD8+ lymphocyte’s in the same group ofAd-mip(P<0.05).Conclusion:Constructed recombinant adenovirus successfully, HEK-293A cell infected by Ad-mip canexpress MIP-1αprotein. And proved that MIP-1αprotein can cause the chemotactic behavior ofCD3+、CD4+、CD8+ lymphocyte and monocyte. Established foundation for the further researchon the function and action mechaniism of MIP-1αin the immune system.

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