| Foot-and-mouth disease virus (FMDV) is the causative agent of the economically most important animal viral disease world-wide. Although mortality associated with FMD is usually low, the disease decreases livestock productivity, and affected countries cannot participate in international trade of animals and animal products. With applications of molecular biology technique to FMDV, FMDV toxic proteases have been reseach hot point.Foot-and-mouth disease virus strain OA/58 RNAs were used as templates for RT-PCR. The amplified cDNA products were cloned into pGEM-T Easy Vectors and transformed into JM109. The Lab gene showed that between two initiation codons, there is a special function sequence which enables small subunit of ribosome to recognize and utilize the second AUG to translate Lpro that is called for Lb; the regions of 35-39th, 43-54th, 65-67th, 75-80th, 90-111th, 113-142th, 144-146th, 148-157th, 159-172th and 176-187th in Lpro most probably are conservative. By homology modeling the FMDV strain OA/58 Lpro, the 3D mold of this protease was obtained. Resolution of the 3D structure of Lpro showed a compact globular form with a flexible C terminal extension from 187th to 201th. Depending on the region of hydrophilicity residues forming hydrophilicity power, Lpro can form dimmers. Lys144, His148 and Asp163 may be active amino acids which form the active site of Lpro. By Ramachandran Plot showing, 3D mold of a FMDV strain OA/58 Lpro is rather reasonable, this research should be used as an instruction in order to direct the work on FMDV Lpro.By the molecular cloning, the Lab gene encoding leader protease called Lpro were cloned in retroviral vector pBPSTR1 to obtain reconstruction retroviral vector termed pBPSTR1-Lab. At different concentrations of puromycin and tetracycline respectively in the cell culture mediums, the growth of bovine kidney cells (MDBK) showed that the optimal puromycin resistant selection concentration was 3μg/mL and tetracycline regulatory concentration was 1μg/mL. Pseudotyped retroviral virus particles were produced by transiently co-tansfecting GP2-293 cells with a retroviral vector DNA and VSV-G plasmid. Then MDBK cells were infected by pseudotyped retroviral virus and were continually seeded in the medium at the optimal tetracycline regulatory concentration and puromycin selection concentration for 12 days to obtain puromycin resistant colonies whose genomes contained the Lab gene. After tetracycline removal, synthesis of Lpro induced severe morphological changes in the puromycin resistant MDBK cells. PCR, RT-PCR and western-blot proved that a stable MDBK cell line inducibly expressing the Lab gene under the control of tetracycline has been obtained.The experiment provides a basis for studying that Lpro of FMDV plays an important role in MDBK cell pathogenesis.
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