| Hand,foot and mouth disease(HFMD)is a kind of acute infectious disease caused by a variety of enterovirus virus,which mainly infected in infants and young children under the age of five.The clinical performance for hand,foot and mouth are rashes in hand,foot,mouth and other parts,seriously as brainstem encephalitis,aseptic meningitis,pulmonary edema and even death.Enterovirus 71(EV71)and Coxsackievirus A 16(CA 16)are the two main pathogens causing HFMD.Previous reports suggest that severe cases of hand,foot and mouth disease are mainly caused by EV71,but a growing number of epidemiological investigation showed that the CA16 caused symptoms are no longer gentle,but also can be severe neurological complications,and even lead to death.Currently,no breakthrough has been achieved in the study of CA16 vaccine,no reports have been reported in clinical trials,and the EV71 vaccine has no protective effect on CA16 infection.Therefore,the CA16 vaccine has important significance for the comprehensive prevention and control of HFMD.The study of live attenuated vaccine is a research direction of CA16 vaccine.Therefore,this thesis mainly aimed at the CA16 live attenuated production process of extraction and purification,in-depth research on the key technology such as the freeze-drying process,and the extraction and purification process and freeze-drying process system evaluation lay the foundation for research and development of freeze-dried live CA16 attenuated vaccine.Chloroform extraction purification process found that PBS washing liquid concentration were 2.0 mmol/L?6.2 mmol/L?10.0 mmol/L,the ratio of the virus and chloroform volume with 1:2,1:1,2:1 and PBS washing liquid washing three or five times to virus infectious titer degree had no significant effect,the mean recovery rate of virus was 81.7%;With the increasing,the number of PBS washing lotion of miscellaneous protein gradually reduce,different virus and chloroform volume ratio and PBS buffer concentration had no obvious effect on the protein removal rate,protein removal rate were greater than 82%,the average removal rate of 88.37%;Different virus and chloroform extraction volume ratio,different PBS washing liquid concentration and PBS washing three or five times had no obvious effect on residual kanamycin content,the results are less than 30 ng/ml,after ultrafiltration concentration,its content is less than 3 ng/ml;The removal rate of residual BSA was 68.87%after chloroform,which was 94.74%after concentration by ultrafiltration.However,the removal rate of the residual chloroform was more than 98%,after concentration by ultrafiltration,which was less than 6 ?g/ml in purification liquid.The result of freeze-drying process shows that the optimal freeze-drying protectant is no gelatin protectant B,and the optimal freeze-drying procedure is program 1 in terms of protectant B.B and freeze-dried procedure one were used to freeze dry.After the freeze-drying process,the product titer was at 5.25 IgCCIDso/ml,and the titer dropped to 1.25 IgCCID50/ml?2.38 1gCCIDso/ml.The product residual moisture test results were 1.29%?1.54%,and the residual chloroform was 7.5 ?g/ml.After immune mice,the mice produced a better humoral immune response,which produced a higher neutralizing antibody,and the GMT was 1:111.43.The results of sensitivity test,abnormal toxicity test and acute toxicity test are all in accordance with the relevant requirements of the?Chinese pharmacopoeia?,indicating that the research of the experimental CA16 freeze-dried vaccine has basically achieved the expected goal.This study obtained the CA16 attenuated vaccine protectant formula,a relatively optimal freeze-drying process and chloroform extraction and purification of CA16 condition,and the drop degree for the freeze-dried products,immunogenicity,allergic experiment and abnormal toxicity experiment and evaluation,the results are in line with the?Chinese pharmacopoeia?,the requirements of the relevant for CA16 attenuated vaccine development laid a solid foundation. |
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