Proliferation Of Transmissible Gastroenteritis Virus Of Swine In PK-15cells Acclimated In Lowserum Medium

TGEV is a highly contagious acute enterovirus,which can cause severe diarrhea,dehydration,vomiting and other symptoms in piglets within two weeks of age,the mortality rate is very high.There is no specific drug or effective treatment for this disease,and its prevention and control is mainly based on injection vaccine.At present,most related vaccine manufacturers use PK-15 cell proliferation TGEV to manufacture TGEV vaccine.Bovine serum is an important nutrient source in the proliferation culture of PK-15 cells,usually reaching about 10% of the medium,but the price of bovine serum is expensive,and each batch of serum changes with the individual differences of the serum cows provided,while the quality of cells cultured with different quality serum has great difference;Secondly,the composition of serum is complex,including cytotoxic substances and inhibitors,which also affect the growth of cells;Bovine serum carries pathogenic organisms such as blood-borne bacteria and mycoplasma,which increases the risk of contaminating cells.In this study,from the actual situation of serum market and vaccine manufacturing,PK-15 cells were domesticated and cultured in a low serum medium,with the conventional medium group(10%FBS)as the control and the low serum medium group(2%FBS)as the experimental group,two different culture media were selected to proliferate and culture TGEV,the virus TCID50 was calculated and the TGEV qRT-PCR assay was established to determine the proliferation of intracellular and extracellular viruses in different media.The results showed that the viral titer of TGEV in the low serum medium was not affected,the intracellular viral content after 24 hours and extracellular viral content after 48 hours in the infected group increased and was significantly higher than that in the control group.The results showed that the PK-15 cells domesticated in this experiment can be used for the proliferation of TGEV.The apoptotic rate of PK-15 cells infected with TGEV in two kinds of culture media was detected by flow cytometry,the results showed that although the concentration of serum in low serum culture system was decreased,but the substitutes for serum have the same effect of delaying apoptosis as serum,and will not accelerate the induction of apoptosis because of the decrease of serum content;After TGEV was infected with PK-15 cells,the apoptotic rate increased with time,the results showed that TGEV could significantly induce apoptosis of PK-15 cells in both media,but there was a significant difference between the two groups at 72 h.It is proved that low serum medium has certain influence on the apoptosis of PK-15 cells induced by TGEV,and how to affect it remains to be further studied;Detect Bcl-xL,Mcl-1 and Puma mRNA transcript levels before and after infection of TGEV with PK-15 cells in two media by the establishment of apoptosis gene Bcl-xL,Mcl-1 and Puma qRT-PCR detection method,it was confirmed that low serum medium has a certain influence on the transcription of apoptotic genes,which affects the occurrence of apoptosis,further research is needed.