in the embodiments described hereinafter , experimental methods in which specific conditions are unspecified are typically carried out under general conditions , e . g . the method described in short protocols in human genetics ( edited by ausubel f . m ., kingston r . e ., seidman j . g . et al ., translated by ma , xuejun , shu , yuelong . beijing : science press , 2004 ). one of the critical prerequisites for developing canine influenza virus vaccines is good seed virus , and the a / canine / zhejiang / 01 / 2010 virus ( h3n2 subtype , zjciv for short ) that is previously isolated in the lab has extremely low hemagglutination titer after amplification no matter on chicken embryos or on cells . thus in the present invention , the primary antigen proteins , i . e . ha and na genes , of the zjciv canine influenza virus , and the other six internal genes of the pr8 virus are recombined together , so as to rescue , by means of the reverse genetic system of the influenza virus , a canine influenza recombinant virus that can generate very high virus titer and hemagglutination titer on both a chicken embryo and a cell , and this recombinant virus can be used as a good seed virus for developing canine influenza vaccines . the total rna of the canine influenza virus zjciv is extracted using trizol ( invitrogen ). with a 12 - bp primer 5 ′- agcaaaagcagg - 3 ′ serving as the specific primer , first - strand cdna is synthesized using reverse transcription system kit ( takara ) according to its instruction . the ha and na of the fragment zjciv are respectively amplified by taking the resultant first - strand cdna as the template and taking sapi - ha - up , sapi - ha - down and sapi - na - up , sapi - na - down as upstream and downstream primers ( containing bspql enzyme digestion sites , as shown in table 1 ). the pcr amplification procedure is as follows : pre - degenerate at 94 ° c . for 5 minutes , enter the following cycle : degenerate at 94 ° c . for 45 seconds , anneal at 53 ° c . for 45 seconds and extend at 72 ° c . for 1 minutes and 45 seconds , complete 30 cycles , and finally , extend at 72 ° c . for 10 minutes . meanwhile , a negative control with no template is established . at the end of reaction , pcr products undergo electrophoresis on 1 . 0 % agarose gel . the results are shown in fig1 : two pcr strips appear , i . e . ha having a size of about 1700 bp and na having a size of about 1400 bp , and destination fragments are consistent in size . in fig1 , m : dna molecular weight marker ; 1 : zjciv ha pcr product ; 2 : zjciv na pcr product . at the end of electrophoresis , the agarose gel of the destination dna fragment is cut off from gel under ultraviolet light , and dna is recovered by a dna rapid recovery kit . the specific method is as follows : cut off the destination - dna - containing agarose gel under a ultraviolet lamp , absorb all the liquid on the surface of the gel by tissue , cut the gel into pieces , put the gel pieces in a sterile 1 . 5 ml ep tube , add buffer de - a ( liquid gel ) the volume of which is 3 times as much as that of the gel ( 100 mg = 100 ul ), mix uniformly and then heat at 75 ° c ., mix intermittently ( for 2 - 3 minutes ) until the blocky gel is completely molten ( about 6 - 8 minutes ); add buffer de - b ( binding buffer ) the volume of which is a half of that of the buffer de - a , and mix uniformly . add isopropanol the volume of which is equal to that of the gel when the recovered dna fragment is less than 400 bp . transfer the mixed solution into a dna preparation tube , centrifuge for 1 minute at a rate of 12000 × g , and pour the waste solution in the collection tube . place the preparation tube back into the collection tube , add 500 ul buffer w1 ( cleaning solution ), centrifuge for 30 seconds at a rate of 12000 × g , and pour the waste solution in the collection tube . place the preparation tube back into the collection tube , add 700 ul buffer w2 ( desalting solution ), centrifuge for 1 minute at a rate of 12000 × g , pour the waste solution in the collection tube , and wash the preparation tube in the same way once again . place the preparation tube back into the collection tube , and centrifuge in vacuum for 1 minute at a rate of 12000 × g . finally , place the preparation tube in the clean 1 . 5 ml ep tube , add 30 ul of de - ionized water to the center of a preparation membrane , stand for 1 minute under room temperature , centrifuge for 1 minute at a rate of 12000 × g to elute dna , and preserve the product at minus 20 ° c . for future use . according to the instruction , the purified pcr products and the pbd vectors ( preserved in the lab ) are incubated at 50 ° c . for 1 hour under the action of bspql restriction endonuclease ( neb ). the enzyme - digested products of the destination fragments and the pbd plasmids are recovered using a gel extraction kit , 1 ul of t4 ligase buffer solution and 1 ul of t4 ligase ( takara ) are added , the reaction system for ligation is 10 ul , and uniform mixing is carried out . ligation is carried out overnight at 16 ° c . the ligation product is transformed to a competent cell jm109 ( prepared in the lab ), and the competent cell is then coated on an amp - containing lb solid culture medium in a clean bench under aseptic conditions , followed by culture for 8 - 20 hours at 37 ° c . a single colony on the lb solid culture medium is picked out and then placed in the test tube having amp - containing lb liquid culture medium that has a volume of about 3 ml , afterwards , the test tube is immobilized on a shaker for shaking culture at 37 ° c . for 10 hours or overnight . the plasmids , which are extracted from the bacterial solution using an alkaline extraction method , are identified by a pcr method . the plasmids identified as positive undergo sequencing , and compared using dnastar sequence analysis software . it turns out that the recombinant plasmids pbd - zjcivha and pbd - zjcivna are constructed successfully , the sequence of the ha gene is shown in seq id no . 3 and the sequence of the na gene is shown in seq id no . 4 . plasmids are extracted using an ultra - pure plasmid extraction kit ( omega ), and the operation steps are as follows : 1 ) dip preserved glycerin bacteria ( containing plasmids pbd - zjcivha , pbd - zjcivna , pbd - pr8m , pbd - pr8pb1 , pbd - pr8pb2 , pbd - pr8pa , pbd - pr8ns and pbd - pr8np , and the last six plasmids are preserved in the lab ) using an inoculating loop , draw lines on the surface of an amp - containing lb flat plate , and stand overnight at 37 ° c . ; 2 ) pick out and inoculate a single colony to a 5 ml amp lb culture medium , carry out shaking culture at 37 ° c . until the od600 value is 1 . 0 - 1 . 5 ; 3 ) collect 3 ml of overnight culture and centrifuge the culture to remove the culture medium thoroughly ; 4 ) bacteria suspension : carry out bacteria suspension using 0 . 25 ml of rnase a - containing solutioni until a homogenate state is reached ; 5 ) cell lysis : add 0 . 25 ml of solutionii , reverse 5 times to achieve gentle and uniform mixing ; 6 ) neutralization : add 0 . 125 ml of buffer n3 , reverse 5 times immediately to achieve gentle and uniform mixing until white flocculent precipitates are formed , and centrifuge for 10 minutes at a rate of 12000 × g under room temperature ; 7 ) carefully pour the supernatant into a clean 1 . 5 ml centrifuge tube , add to the supernatant an etr solution ( blue ) the volume of which is one - tenth of that of the centrifuge tube , reverse 7 - 10 times , and put in an ice bath for 10 minutes ; 8 ) add a water bath at 42 ° c . for 5 minutes , then mix to reach a turbid state , and centrifuge for 3 minutes at a rate of 12000 × g under room temperature , so as to form a blue layer in the etr solution at the bottom of the centrifuge tube ; 9 ) transfer the supernatant into a new 1 . 5 ml centrifuge tube , add absolute ethyl alcohol the volume of which is a half of that of the centrifuge tube , reverse 6 - 7 times , and stand for 1 - 2 minutes under room temperature ; 10 ) pour the aforementioned mixed solution into a hiband dnamini column that is properly balanced by 2ml of e4 in advance , put the column on a 2 ml collection tube , and centrifuge for 1 minute at a rate of 10000 × g under room temperature so that lysis solution passes by the column ; 11 ) discard the liquid in the collection tube , add the remaining mixed solution into the column , and centrifuge for 1 minute at a rate of 10000 × g under room temperature so that lysis solution totally passes through the column ; add 500 ul of buffer hb to the column , centrifuge for 1 minute at a rate of 10000 × g under room temperature , and wash the column to ensure removal of the residual proteins , in order to obtain high - quality dna ; 12 ) discard the liquid , wash the column with 700 ul of dna wash buffer , centrifuge for 1 minute at a rate of 10000 × g under room temperature , and discard the liquid ; 13 ) repeat the operation and then add dna wash buffer ; 14 ) discard the liquid , carry out idling , centrifuge for 2 minutes at a rate of 12000 × g under room temperature , and discard the liquid ; 15 ) put the column in a clean 1 . 5 ml centrifuge tube , add 30 - 50 ul of endotoxin - free eluant onto the column , stand for 2 minutes under room temperature , and centrifuge for 1 minute at a rate of 12000 × g under room temperature to elute dna , wherein this elution can be carried out twice ; and 16 ) carry out electrophoresis detection , and measure od 260 and od 280 using an nanodrop 2000c ultraviolet - visible spectrophotometer to estimate dna content and purity . results : a sufficient amount of plasmids required by transfection are acquired . the aforementioned plasmids extracted at a ultra - purity , including pbd - zjcivha , pbd - zjcivna , pbd - pr8m , pbd - pr8pb1 , pbd - pr8pb2 , pbd - pr8pa , pbd - pr8ns and pbd - pr8np , are co - transfected via a proper amount of liposome 2000 to a 293t cell having a diameter of 3 . 5 cm . 6 hours after transfection , the cell supernatant is discarded , 2 ml of opti - mem ( invitrogen ) culture solution is added , and the cell is put in a co 2 incubator at 37 ° c . for culture for 72 hours . 48 hours after transfection , the cell supernatant is inoculated to a 9 - day to 11 - day spf chicken embryo ( beijing merial vital laboratory animal technology co ., ltd . ), the chicken embryo is sealed by paraffin and then put in an incubator at 37 ° c . for culture . 48 to 72 hours later , the chicken embryo undergoes a temperature of 4 ° c . overnight , and then taken out to obtain chicken embryo allantoic fluid . the presence of hemagglutinin activity in the allantoic fluid is determined by a hemagglutination test . results : hemagglutination occurs in the allantoic fluid , indicating that a pr8 recombinant virus containing the ha and na genes of the zjciv has been rescued successfully . the total rna of the allantoic fluid of the recombinant virus is extracted using trizol , and then subjected to reverse transcription by a 12 - bp primer to obtain first - strand cdna . the ha and na fragments of the zjciv are pcr - amplified by taking the first - strand cdna as the template and taking sapi - ha - up , sapi - ha - down and sapi - na - up , sapi - na - down as upstream and downstream primers , and then identified by 1 % agarose electrophoresis . in addition , the zjciv ha and zjciv na pcr products undergo sequencing in the company . results : on agarose electrophoresis gel , there are two strips having a size of about 1700 bp and a size of about 1400 bp respectively , which are completely consistent with the target sizes . and it is further proved by sequencing results that these pcr products are indeed the ha and na fragments of the zjciv . 1 . determination of eid 50 of the rescued recombinant virus and the zjciv the virus - containing chicken embryo allantoic fluid undergoes 10 - fold dilutions , and the chicken embryo allantoic fluids that are diluted based on the dilutabilities from 10 − 6 to 10 − 10 are respectively inoculated to five 9 - day to 11 - day spf chicken embryos for continuous incubation for 48 hours at 37 ° c . whether the chicken embryo allantoic fluids are infected is judged by determining the hemagglutinin activities of the infected embryo allantoic fluids , and eid 50 ( chicken embryos &# 39 ; median infective doses ) is calculated using a reed - muench method . results : eid 50 of the rescued recombinant virus and the zjciv are 10 7 . 5 / 100 ul and 10 6 . 5 / 100 ul , respectively . 2 . determination of tcid 50 of the rescued recombinant virus and the zjciv 10 - fold dilutions start from 1 : 10 − 3 , the recombinant virus and zjciv having different dilutabilities are inoculated to a 48 - well plate on which monolayer mdck cells grow , and the inoculation procedure is as follows : at first , clean the mdck cells twice with pbs , then add 100 ul of virus to each well , repeat this operation 3 times for every dilutability , put the 48 - well plate in a co 2 incubator at 37 ° c . for the purpose that viruses are adsorbed on the cells , shake the cells left and right once at an interval of 20 minutes , discard the viruses in the cell culture plate 1 . 5 - 2 . 5 hours later , wash the cells twice with pbs , and then add 300 ul of serum - free medium . continuously culture the virus - adsorbed cells in the co 2 incubator for 72 hours , then determine the hemagglutinin activity of every well , and calculate tcid 50 ( tissue cells &# 39 ; median infective doses ) using a reed - muench method . results : tcid 50 of the rescued recombinant virus and the zjciv are 10 6 . 5 / 100 ul and 10 5 . 5 / 100 ul , respectively . 3 . growth characteristic comparison of the rescued recombinant virus and the zjciv on the chicken embryos the rescued recombinant virus and the zjciv are diluted as 100eid 50 , and are inoculated , based on this dilutability , to 18 9 - day to 11 - day spf chicken embryos , respectively . 6 hours , 12 hours , 24 hours , 36 hours , 48 hours and 72 hours after inoculation , 3 inoculated spf chicken embryos are taken out respectively , their allantoic fluids are collected and the hemagglutinin activities thereof are determined ( fig2 ). the allantoic fluids collected at different time undergo 10 - fold dilutions , the virus solution of each dilutability is inoculated to 3 9 - day to 11 - day spf chicken embryos , and the inoculum dose is 100 ul per chicken embryo . 48 hours after inoculation , the hemagglutinin activities of the chicken embryo allantoic fluids are determined , the virus contents of the allantoic fluids collected at different time are calculated , a growth curve of the virus is drawn up ( fig3 ), and the growth conditions of the rescued recombinant virus and the zjciv on the chicken embryos are compared . the results are shown in fig2 : the allantoic fluids of both the zjciv and the recombinant virus have no hemagglutination within 12 hours after inoculation . 48 hours after inoculation , the hemagglutination titer of the recombinant virus reaches 2 11 , which is significantly higher than that of the zjciv . the results are shown in fig3 : no virus is detected 6 hours after inoculation of the zjciv and the rescued recombinant virus . 12 hours after inoculation , the virus titer of the rescued recombinant virus is significantly higher than that of the zjciv , and reaches the peak 48 hours after inoculation . 4 . growth characteristic comparison of the rescued recombinant virus and the zjciv on the mdck cells the rescued recombinant virus and the zjciv are diluted as 100tcid 50 , and are each inoculated , based on this dilutability , to 3 t25 cell bottles in which 80 % of the mdck cells grow . 6 hours , 12 hours , 24 hours , 36 hours , 48 hours and 72 hours after inoculation , their cell supernatants are collected respectively and the hemagglutination titers thereof are determined ( fig4 ). the virus contents in the cell supernatants collected at different time are titrated , specifically as follows : the collected cell supernatants undergo 10 - fold dilutions , the virus solution of each dilutability is inoculated to the mdck cells with 80 % growing in 3 wells of a 24 - well cell plate , the hemagglutinin activities are determined 48 hours after inoculation , the virus contents ( tcid 50 ) in the cell supernatants collected at different time are calculated , a growth curve of the virus is drawn up according to this tcid 50 ( fig5 ), and the growth conditions of the rescued recombinant virus and the zjciv on the mdck cells are compared . the hemagglutination titer results are shown in fig4 : within 12 hours after inoculation , the cell supernatant of the zjciv has no hemagglutinin activity , while the cell supernatant of the recombinant virus has hemagglutinin activity , which reaches the peak 36 hours after inoculation and subsequently tends to a relatively stable state . in the entire infection process , the hemagglutinin activity of the recombinant virus is significantly higher than that of the zjciv . the virus titer results are shown in fig5 : no virus is detected 6 hours after inoculation of the zjciv and the rescued recombinant virus , and 12 hours after inoculation , the virus of the rescued recombinant virus can be detected , whereas the virus of the zjciv cannot be detected until 24 hours after inoculation . 36 hours after inoculation , the virus titers of the recombinant virus and the zjciv reach the peak and then begin descending . in the entire cell infection process , the virus titer of the recombinant virus is significantly higher than that of the zjciv . the embodiments described above are for illustrating the ways of practicing the present invention only , and shall not be understood as limiting the patent scope of the present invention even if their descriptions are specific and detailed . it shall be noted that , many modifications and improvements could also be made by those ordinary skilled in this art without departing from the concept of the present invention , and these modifications and improvements shall fall within the scope of the present invention . accordingly , the patent scope of the present invention shall be subject to the claims appended .
a canine influenza recombinant virus includes ha and na genes of zjciv canine influenza virus as well as six internal genes pa , pb1 , pb2 , m , np and ns of a pr8 virus . the nucleotide sequence of the ha gene is selected from the group consisting of : a nucleotide sequence encoding an amino acid sequence of seq id no . 1 ; a nucleotide sequence encoding an amino acid sequence which has at least 98 % sequence identity to the amino acid sequence of seq id no . 1 . the nucleotide sequence of the na gene is selected from the group consisting of : a nucleotide sequence encoding an amino acid sequence of seq id no . 2 ; a nucleotide sequence encoding an amino acid sequence which has at least 98 % sequence identity to the amino acid sequence of seq id no . 2 .