Expression Of Norovirus VP1 Protein Genein Pichia Pastoris And The Product Analysis

Background and Objective: Human norovirus (NoVs) are a major cause of epidemic and sporadic gastroenteritis worldwide, and VLPs have recently been explored as potential vaccine platforms due to the high degree of immunogenicity. Expression of the main structural protein, VP1, leads to formation of self-assembled particles with similar characteristics to the original virus. The objective of this study is to construct the recombinant plasmid pPIC9K-VP1 with Viral RNA for secretory expression of the norovirus capsid protein efficiently in Pichia pastoris. These production and purification systems described can be used for large-scale production of Norovirus virus-like particles as vaccine platform candidates.Methods: From Dec 2015 to Aug 2016, specimens and clinical data were collected from adult patients with acute nonbacterial enteritis at the department of gastroenterology, General Hospital of Chinese Navy. Samples were converted to suspensions and centrifuged, and Virus RNA was extracted from the supernatant.Extracted viral RNA was amplified with RT-PCR and sequenced. We then selected samples of NoVs-positive and get the target gene. To construct pPIC9K-VP 1 with the cDNA-NoVs as template, the gene sequence of VP1 protein was inserted into pPIC9K. Recombinant plasmid pPIC9K-VP1 was linearized by Bam H I, Sal I and transformed into yeast host strain Pichia pastoris GS115 by electroporation. Glycerol is added to the culture medium in portions at the initial stage of fermentation. After depletion of glycerol, induction of NoVs-VLPs production was initiated by the addition of methanol. The supernatant from the shake flask culture was collected by centrifugalization, concentrated by ultrafiltration, purified via ion-exchange chromatography, desalted by dialysis and assayed by SDS-PAGE, TEM and Western blot.Results: A total of 43 fecal specimens and clinical data were collected from patients.The conservative fragment of VP1 protein encoding gene was amplified with RT-PCR and sequenced. The gene sequence is about 1620 bases. Analysis through online BLAST,the strain represented by the sequence obtained was assigned to NoVs G?-4.NoVs-VP1 protein gene had been successfully cloned into expression vector PIC9K,and VLP sprotein was secreted into fermentation supernatant. The SDS-PAGE gel showed a band at 62 ku corresponding to the molecular weight of the VLPs protein.Western Blot using the anti-NoVs serum also revealed a positive band at 62 ku confirming the expression of the full-length VP1 protein, and the yield of VLPs reached 1 g/L (about 600 mg/L after purification) . The morphology and size of the NoVs-VLPs were also studied using transmission electron microscopy, confirming fully formed particles, with an average size of 40 nm.Conclusion: In this study we report a method for the production of fully assembled NoVs-VLPs with high yield from Pichia pastoris. The NoVs-VLPs were produced using a bioreactor and purified directly from the culture medium, via ion-exchange chromatography, with a final purity product over 90%. These production and purification systems described can be used for large-scale production of Norovirus virus-like particles as vaccine platform candidates.