Establishment Of Long-distance RT-PCR Method Of IBVZZ2004 Strain Isolated From Duck And Construction Of The Full-length CDNA Library

Infectious bronchitis virus strain(IBV ZZ2004)isolated from duck was an important virus that mainly causes the suppression of growth and immunity on poultry.Based on the RT-PCR technology and complete genome sequence analysis,this research established the long-distance RT-PCR method of 1BVZZ2004 strain and constructed the cDNA library.It lays the foundation for the further reverse genetics technology research about the virus.Based on the complete genome sequence of IBV ZZ2004 strain,they were designed the F1/F2 pair of specific primers for 20370?24541 nt,which includes S,3a,3b,and E genes.Throuth the heat-resistant DNA polymerase with 3'-5'Exonuclease activity,amplifications were respectively used through 2Step method and 3Step method,primer concentration,annealing temperature,extended time were optimized,under the optimized conditions,the 3Step method in order to primer concentration is 20pmol/?L,the annealing temperature of 61?,circulation 30 times for the best reaction condition;2Step method in order to primer concentration is 20pmol/?L,the annealing temperature of 68 ?,extended time of 3 min,circulation 30 times for the best reaction condition.Compared with the3Step method,2Step method is simple and quick,therefore,2Step method is the long distance RT-PCR of the most appropriate method.With this improvement,gene fragment size of 4171 bp was amplified,which was consistent to the expected target fragment size,and a long distance RT-PCR method was also successfully established.By means of the complete genome sequence of IBV ZZ2004 strain and DNA Star software analysis,a single restriction enzyme cutting site of the full-length genome nucleotide sequence was selected,and 13 pairs of primer were prepared.Using RT-PCR technology,13 overlapping cDNA fragments of F1?F13 containing the ZZ2004 virus genome were successfully amplified,which were then cloned into the pMD-19T vector,respectively.13 different recombinant plasmid get,Using JM109 competent cell chemical conversion,Through the PCR and sequencing identification,IBV ZZ2004 strain cDNA library,including 13 cDNA fragments,was established.In addition,4 pairs of primer were designed and synthesised,with the use of the established long-distance RT-PCR method,4 overlapping cDNA fragments of N1?N4 containing the ZZ2004 virus genome were successfully amplified,they were Nl(1nt?5954nt)?N2(5860nt?12491nt)?N3(12434nt?20930nt)?N4(20717nt?27673nt),which were then cloned into pGEM-T easy vector,respectively.4 different recombinant plasmid get,Using DH 5a competent cell chemical conversion,through the double digestion and sequencing identification,IBV ZZ2004 strain cDNA library,including 4 cDNA fragments,was established.Then,the two overlapping fragments in four fragments were connected,which that IBV fragment and N2 fragment were connected by Bsp1191 enzyme,the N3 fragment and N4 fragment were connected by Swal enzyme,The length are respectively 12456bp and 15217bp.and then,which were cloned into pFastBacHTA vector respectively,lt was formed IBV ZZ2004 strain cDNA library only including only two fragments,It lays the foundation for the construction of the full-length cDNA.In conclusion,this research successfully constructed the long distance RT-PCR method of IBV ZZ2004 strain.Using the long distance RT-PCR method,the cDNA library was constructed,which contains fewer cDNA molecular cluster,and therefore more easily to construct the full-length cDNA molecular.Compared with the cDNA library constructed with the ordinary RT-PCR technology,our method was more convenient and quickly.