Construction of recombinant pLNCX/iNOS and titer assay in NIH3T3 cells

Objective To construct the recombinant pLNCX/iNOS.Methods Full length cDNA of iNOS were amplified by PCR from pCMV/iNOS vector with retroviral vectors to construct pLNCXiNOS.Purified recombinant retroviral vectors DNA were transferred into the amphotropic packaging cell line PA317 by means of liposome mediated method and screened anti?G418 positive clones which were cultured?abstracted superant liquid for further concentration.The viral titer was determined with the NIH3T3.Results The gene rank of iNOScDNA amplified by PCR was coincidence with its gene map through enzyme cutting and gene sequencing analysis.16 anti?G418 positive clones can stably synthesis and excrete recombinant retroviral vectors.The titer of recombinant retroviral vector was 2.1×10 4CFU·ml -1 .Conclusion The iNOS gene can be effectively transferred by retroviral vector pLNCX.