Swine Pseudorabies Vaccine Of Live Thermo-stable And Technology In Controlling Freeze-drying

In this study,the swine pseudorabies virus strains used to produce vaccine was theHB2000strains, which was constructed by Huazhong Agricultural University utilizingthe gene knockout technology and making the gene of TK、gE、gI of PRV strain Amissing, namely the PRV Ea TK-/gE-/gp63-strains (gI gene) gene mutations. Itwas acquired in2000,successful,named HB2000strains.The result of virusneutralization tests show that the venom of HB2000strains could be neutralized bytype-specific serologic of PRV strain A;the result of PCR demonstrated that the specificgene segments of748bp TK and514bp gE-gI could be successfully amplified,indicating a good specificity of virus strains.The response surface method (RSM) was employed to optimize the formula ofheat-resisting protective agent of the vaccine.The results showed that sodium glutamate,L-arginine hydrochloride and D-sorbitol can significantly improve the tolerance abilityof virus under freeze-dried environment,while the significances decreased from enzymehydrolysed casein, trehalose,gelatin, sucrose to BSA;Taking virus protection rate afterfreeze-drying as the response value,the optimum formula were:glutamate2%,L-arginine hydrochloride2%,D-sorbitol2%,the enzyme hydrolysed casein7.6%,trehalose1%,gelatin4%,sugar15.2%and BSA2%.Validation experiments of theformula were also carried out,showing that the protection rate after freeze-drying couldreach80.40%.Furthermore,the virus droplets obtained by keeping the vaccine at37℃for7days for the aging resistance test dropped0.55,0.62,and0.47while the ones ofvaccine stored at2~8℃for24months dropped0.47,0.51, and0.41,indicating that thevaccine which with the optimum formula had a good thermal stability.On the basis of cryopreserved test and melting point determination results,thefreeze-drying parameters were systematically investigated.The optimum process wasobtained as follows:①precool stage: the product was cooled under-42℃for3h afterthe temperature of plates dropped to-42℃within1h;②sublimation drying stage: thetemperature of plates was increased from-42℃to0℃within2h and lasted for15h;③parsing drying stage: the temperature of plates was increased from0℃to28℃within2h and lasted for8h;④the end of the freeze-drying.The whole freeze-dryingprocess lasted for31h.The resulting products exhibit a milky white loose spongy conglomeration state,5/5purple glow,residual water percentage of1.7%~2.2%,survivalrate of88.73%.Moreover,the loss of virus droplet at37℃for7days was less than1and the product could be stored at2~8℃for24months,which is consistent with currentstandards.Safety and efficacy test was performed suing pigs.The results show the same5/5security among the groups of single dose inoculation,single dose repeat inoculation andoverdosage inoculation,which demonstrates no significant difference to the normalpiglets without treatment.The animal immune attack drug test displays that the controlgroups of tapping poison have5/5morbidity while groups of vaccination achieves5/5protection.Also the results of virus content in vaccine and the poison attack protectioncorrelation show that after poison protection groups of103.0TCID50inoculation achieve0/5protection;groups of104.0TCID50inoculation achieve3/5protection and groups of105.0TCID50and106.0TCID50inoculation both receive4/5and more than thatprotected.The control groups of poison attack with5/5immune protection exhibittypical clinical symptoms of porcine pseudorabies, such as continuous highfever,dyspnea and neurological symptoms. On the other hand,for the pregnant sows,the groups of poison attack show4/5morbidity and they gave birth to piglets ofstillbirth,mummy foetus, and weak young piglets. Compared with the2/5protectionrate of104.0TCID50inoculation groups, the groups of105.0TCID50and106.0TCID50inoculation exhibit4/5and more than that of sows could be protected.It indicated thatvirus content in vaccine have a positive correlation with the poison attack protectionrate and the later increased with the rise of the former. Therefore the minimum immunedose for piglet and sows will be determined as105.0TCID50.Neutralizing antibody titerwas a positive correlation with poison attack protection,when piglets neutralizingantibody titer is greater than or equal to1:16, neutralizing antibody titer is greater thanor equal to1:12,100%of the piglets and sows can get poison protection. Achieved thequality standards of similar imported products.