Recombinant Herpes Simplex Virus Type Ⅱ Genome Stability Study

Objective To regonize the stability of OH2 genome,provide evidence for further study.Methods The experiments included DNA extraction,PCR product restrict-digestion that identified herpes simplex virus,PCR validation of OH2 gene modification,cell culture and ELISA detection of hGM-CSF levels.The HSV-1 and HSV-2 are distinguished by restriction fragments after PCR amplification.This experiment is designed based on g D glycoprotein conserved sequence.HSV-1 has a Msp I enzyme site on the conserved sequence while HSV-2 does not have.Using a pair of primers derived from the g D glycoprotein conserved sequence,a 200 bp fragment can be amplified from both HSV-1 and HSV-2.However,when digested with Msp I enzyme,HSV-1 can get two fragments about 130 bp and 70 bp while HSV-2 can only get one fragment about 200 bp.This test can identify OH2 without contamination of HSV-1 that is widely presented in human population.The OH2 modifications can be identified by PCR/agrose gel analysis with the primers designed from the sequences for deletion,insertion and down/up-stream flanking regions.hGM-CSF expression in OH2 culture supernatant can be detected by ELISA.The purpose of the experiment is to determine whether the hGM-CSF gene inserted in the OH2 genome was consistently expressed.Using coated mouse MAb specific to hGM-CSF and second antibody(hGM-CSF Ab-biotin)in the experiment,the OD value was measured after incubation.The standard curve was created with the horizontal axis for the logarithm of hGM-CSF standard concentration and with the vertical axis for the OD value(sample OD value minus blank OD value).The tested sample was diluted with appropriate dilution factor(about 40-80 times),so that the OD value could fall in the linear scope of the standard curve.The sample OD value was brought into the standard curve to obtain hGM-CSF concentration,followed by multiplication of the dilution factor to get the sample hGM-CSF content.The results obtained from a series of tests indicated that the hGM-CSF content in the samples was generally more than 2 ng/ml.Result The HSV-1 has two fragments of 130 bp and 70 bp by enzyme digestion in the identification experiment,while HSV-2 and OH2 have only one fragment of 200 bp.With OH2 viral DNA the correct size fragments can be amplified by the four pairs of primers in the genetic modification indetification experiments.The liner scope of hGM-CSF standard curve is between range 31.25-500pg/ml,and the expression of hGM-CSF is generally more than 2ng/ml in OH2 culture supernatant.Conclusion The modified genes in OH2 virus genome are stable,and the hGM-CSF expression can be detected.