Construction Of The Eukaryotic Expression Recombinant Plasmid Containing NDV HN Gene

In this study five recombinant plasmids containing HN (hemagglutinin-neuraminidase) genes of NDV (Newcastle disease virus) were constructed,HN genes were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) and were inserted directly into eukaryotic expression plasmid pcDNA3.Two NDV strains,Lasota and F48E9,were propagated in 9-day-old embryonated specific-pathogen-free(SPF) eggs respectively for 36 hours at 37,then these chicken embryos were placed at 4 for 12h before the allantoic fluid was gained. According to HN gene sequences of both NDV strains registered in GenBank and multi-clone restriction enzyme sites of eukaryotic expression plasmid pcDNA3,we designed two pairs of special primers,namely PI:5 -CCCAAGCTTATGGACCGCGCCGTTAGCCAAG-3 ,P2:5 -GCTCTAG-ACTAGCCAGACCTGGCTTCTC-3 andP3:5 -CGGGATCCATGGACCG-TGTAGTTAGCC-3 ,P4:5 -CCGCTCGAGTTAAATCCCATCATCCTTG-AG -3 o With them,the HN genes of both strains were amplified by RT-PCR respectively. A DNA band about 1700 bp in length was found in the RT-PCR products of both strains after an agarose-gel electrophoresis,which was consistent with the length of HN registered in GenBank. Further these HN gene cDNAs were inserted directly into pcDNA3 vectors respectively and their products were identified by double endonucleases digesting. Four recombinant plasmids,pcDNA3-LHN1 pcDNA3-LHN2 pcDNA3-LHN3 and pcDNA3-LHN4,containing HN genes of Lasota and one,pcDNA3-FHN,containing HN gene of F48E9 were obtained. Determination of cDNA sequences of HN in pcDNA3- LHNl and pcDNA3 -FHN were carried out,the results showed both HN gene sequences were consistent with the corresponding sequences in GenBank.In a word,we had successfully constructed several recombinant plasmids containing HN genes of strain,Lasota or F48E9,which would provide a possibility for further applying the recombinant plasmids as DNA vaccine.