Authentication Of Cells For Human Vaccine Production And Establishment Of A PCR Assay For Porcine Circovirus And Torque Teno Sus Virus Strains In Vaccine-related Cell Lines

Cell substrate is the main carrier of vaccine production and quality control including primary cells,diploid cells and continuous cell lines.Now there are human diploid cell line(KMB17 cell)and continuous cell line(Vero cell)used for vaccine production in Institute of Medical Biology.The safety of cell substrates is closely related to the safety of the final product.Cell substrates should be tested thoroughly including cell identification and extraneous agent when they are used for the production and verification.Karyotype analysis is used for cell identification in our institute.This method can identify human cells.However human diploid cells KMB17 and MRC-5 cannot be identified because of its same chromosome characteristics.Short tandem repeat(STR)markers are highly polymorphic among human population.And they have been widely used in paternity test.Because of simple operation,strong specificity,high sensitivity,STR-PCR method is one of the simplest and most effective methods for cell identification and cross contamination.For the shortcomings of the Karyotype analysis in cell substrates,STR method is established for cell identification.The technology of short tandem repeat(STR)-PCR method was used to sequence nineteen autosomal loci 19S433,D5S818,D21 S11,D18S51,D6S1043,D3S1358,D13S317,D7S820,D16S539,CSF1PO,PentaD,vWA,D8S1179,TPOX,Penta E,TH01,D12S391,D2S1338,FGA and one sex chromosome locus AMEL in KMB17 cell line.The profiles are identical determined by two times of repetitive DNA profiling.Compared with sixteen chromosome loci reported by Meng Shufang from National Institute for Food and Drug Control,the results of these sixteen STR profiles are exactly the same.It proves that our sample is KMB17cell.The detection procedure and results are feasible and reliable.According to study of Almeida from National Institute of Standards and Technology the eight loci D8S1106,D1S518,D6S1017,D17S1304,D4S2408,D5S1467,D19S245 and DYS389 respectively in Vero cell line were used to sequence by STR-PCR method.The profile of the Vero cell line and the number of repeats are identical compared with the research results from Almeida et al.It proved that Vero cell for vaccine production is the correct cell lines,and is not contaminated.Testing of the operation process is repeatable,accurate and reliable.Adventitious virus agent is an important factor that affects the security of cell substrate.Detection of porcine-origin virus is added to preparation of animal cell substrate and quality control in the biological products of production and quality control,section three,2015 edition of“Chinese pharmacopoeia".Polymerase Chain Reaction(PCR)is a kind of method with strong specificity,high sensitivity,simple operation,timesaving,etc.It can expand specific DNA fragments rapidly in vitro.PCV(porcine circovirus)and TTSuV(Torque teno sus virus)synthetic genome sequences were inserted into PUC57 carrier plasmid as positive control.The temperatures for annealing in PCR assay were optimized.The optimized PCR assay was verified for sensitivity and specificity,and used for tests for PCV and TTSuV in raw material(for example,trypsin),cell substrates(including KMB17,Vero,MRC-5,Hep2,BT),virus harvests and final products of vaccine.Results showed that there were no porcine DNA fragments in test samples.Specificity:PCV1 and PCV2,Parvovirus(PPV),TTSuV 1 and TTSuV2 plasmids are used as control,and specific DNA bands were amplified by the developed PCR assay only from the genomes of specific templates,other plasmids showed no PCR amplification.Sensitivity:PCV and TTSuV recombinant plasmids will be diluted to the concentration of 10ng/ul,1ng/ul,100 pg/ul,10pg/ul,1pg/ul,100fg/ul,10fg/ul,1fg/ul,0.1fg/ul,0.01fg/ul.With its as a template for PCR amplification,2%agarose gel electrophoresis was used to detect PCR product.The minimum detection limit of the developed PCR assay of PCV was 0.1 fg,and is equal to1.34× 104 copies of PCV.The minimum detection limit of the developed PCR assay of TTSuV 1 and TTSuV2 were 10fg and 0.0 1fg respectively.And equal to 1.92 × 106 copies of TTSuV1 and 1.94 ×103 copies of TTSuV2.These results show that the developed PCR method has the specificity and sensitivity,and can be used for my vaccine production and quality control.