The Detection Of HBsAg Encoding Plasmid Copy Number In Recombinant Yeast By Real-time PCR

The genetic recombinant yeast, Saccharomyces cerevisiae 2150-2-3, carrying the plasmid(pHBS56-GAP347/33)~1 contains the region encoding the hepatitis B virus surface antigen (HBsAg) is employed to manufacture the recombinant hepatitis B vaccine. The current control test for the plasmid stability in the yeast is selective replica plating method, which can only test the plasmid maintaining percentage in the yeast cells, but cannot test the real copy numbers in yeast cells. Generally, the recombinant protein expression level is determined by the amount of the plasmids which encode the target protein, so the plasmid copy number is the key parameter needs to be detected during production process. Several techniques could be used to test the plasmid copy number, such as CsCl-EB density gradient centrifugation, HPLC , Southern blot etc. In this reasearch, an assay for the plasmid copy number in the host yeast cells was established, which using a novel real-time polymerase chain reaction (real-time PCR) techniques to detect the quantities of HBsAg gene within the plasmid and a unique single copy URA3 gene in the host genome as intra-reference simultaneously, the plasmid copy numbers for each cell could be deduced conveniently without preparing usual reference sample and calculating host cell numbers. The fermentation harvest sample was tested for the plasmid pHBS56-GAP347/33 copies with the assay, the copy number was 71.5 copy/cell, the test was also verified by Dot-blotting assay. The method has good accuracy and the whole measure procedure can be done in 90 minutes, whereas the replica plating test lasts for 5 days.This article also described the application of the real-time PCR. 16 sub-clones of Saccharomyces cerevisiae 2150-2-3 were selected and incubated in shaking flasks for 3 successive passages. To survey the relationship between the plasmid copy number and HBsAg expression level, the two parameters were detected for eachpassage, and the data showed that the variation of plasmid copy numbers for different sub-clones were great and the two parameters were obviously correlated. The three lots of laboratory shaking flask and two lots of plant manufacturing fermenter experimental fed-batch fermentation for the recombinant yeast were carried out for longer time other than regular 40 hours production fermentation, the samples at 40,52 and 64 hours were collected for plasmid copy number assay by the real-time PCR. The results were 30.68 > 38^ 43.06 and 85^ 86^ 82.5 respectively, which implied that prolonging the ferment time could increase the cell density and protein expression level and did not influence the stability of the plasmid. The plasmid copy number detected for the three lots of batch ferment in shake-flask and three lots of plant seed fermenter as well as manufacturing fermenter harvests provided the data to evaluate the status of the fermentation process during production.