Clone Of Hantaan Virus M2 Gene And The Expression Of The Protein

ObjectiveHemorrhagic fever with renal syndrome, (HFRS) is a acute viral infectious disease with the character of acute onset, severe symptoms and high mortality, which is caused directly by the Hantaan virus and currently we do not have effective therapeutic methods. HFRS is caused by the virus belonged to Hantaviras, Bunyaviridae. Based on serological and molecular biological methods, Hantaviras could be divided into Hantaan virus, Seroul virus, Puumala virus, Prospect hill virus and others. 76-118 strain is a typical virus of Hantaan virus. Research results revealed that the envelop glycoprotein (G1, G2) of Hantaan virus could stimulate the organism to produce neutralizing antibodies, which could protect the infected animals and human from being attacked by the virus, and the neutralizing antigen epitope of the glycoprotein mainly located on G2. This experiment aims at constructing a prokaryotic expression vector of Hantaan virus M2 gene to harvest the G2 protein, which provides the basis for anti-Hantaan virus antibody preparation and screening antibody library anti-Hantaan virus antibodies.With this purpose, our study carried out the following projects: (1) Using plasmid pGEX-6P-l as vector, we constructed the prokaryotic recombinant expression plasmid of pGEX-6P-1/M2; (2) Expressing the fusion protein GST-G2 in E.coli. strain BL21.MethodsI. Clone of M2 geneUsing Hantaan virus M as the template and undertook PCR reaction which amplified the 16,000 bp DNA fragments containing the complete Hantaan virus G2 protein encoding gene M2. Performing the subsequent gel recover of the Hantaan virus M2 fragments and stored the products in - 20 deg C.II. Construction of PMD18-M2 plasmidEndonuclease digesting the PCR products and T vector pMD18 with Xho I and BamH I, and then ligating the products at 16 deg C for approximately 16 hours under the catalyzing of DNA ligase. According to the instruction of Molecular Cloning, transforming 10μl ligated products into the competent E. Coli. DH5α. After that, spreading the post-transformed DH5a. on the LB plate containing 50μg/mL ampicillin and ensuing incubating at 37 deg C incubator for 16 hours. Subsequently, picking up a monoclonal colony and amplifying the colonies for future recombinant plasmids extraction with alkaline lysis method. Finally, store the extracted plasmids in - 20 deg C.III.Construction of prokaryotic expression vector of pGEX-6P-l/M2Double endonuclease digesting of the plasmids acquired in last step and pGEX-6P-1 with BamUI and XhoI and subsequently, ligating the products with T4 DNA ligase at 16 deg C for approximately 16 hours. After ligation, transform 10μl products into competent E. Coli. DH5α, and spread the transformed DH5αon the LB plate and ensuing incubating at 37 deg C incubator for 16 hours.IV. Inducted expression and purification of GST-G2 fusion proteinInnoculate E. Coli. BL21 containing the pGEX-6P-1/M2 plasmids into LB broth plus ampicillin and then shaking culture over night at 37 deg C. The next day, transfer the BL 21 containing LB broth into fresh Amp⁺ LB broth with the diluting ratio of 1:50. Then, put the latter one at 30 deg C incubator continuing shaking culture until the mid-point of logarithmic phase has been reached. When the OD₆₀₀ value of the culture broth reached to 0.5～0.6, add IPTG to the final concentration of 0.1 mmol/L, the culture broth without IPTG was set as negative control and continue 30 deg C shaking culture for 3-4 hours. After 3 hours' culture, take out 1 ml bacterial containing broth and transfer to a fresh EP tube, centrifuge and subsequently discard the supernatant. Examine the expression of GST-G2 protein using SDS-PAGE method and optimize the expression condition for large scale amplifying induction. Centrifuge at 7,700X g for 10 min at 4 deg C to collect the bacteria, and then re-suspend the bacteria with 50 ml pre-chilled PBS. After that, add bacterial lysis buffer with ensuing ultrasonic lysis, then centrifuge at 12,000g for 10 min at 4 deg C and take out the supernatant for Glutathione Sepharose 4B purification of GST-G2 protein according to the manual.ResultsI. Amplification of M2 gene fragmentsAfter the PCR amplification, a specific 16,000 bp band could be observed on agarose gel which coordinates with the expectation.II. Endonuclease digestion identification of the pMD18-M2recombinant plasmidDouble endonuclease digestion of the plasmid with EcoKV and BamHI, then identifying the product on 1% agarose gel electrophoresis and the gel image could be used to prove that the plasmid containing the right gene. The plasmids had been sequenced by Peking Sanboyuanzhi Biocompany and the sequence had been proofread with the data of M2 gene in GenBank database, the homogeneity reached to 98.8% and the opening reading frame was correct.III. The induced expression and purification of the recombinantbacteria containing pGEX-6P-1/M2.The sequencing proved GST-G2 fusion gene expression vector E.coli BL21 is the expression strain of GST-G2 fusion protein. Taking out IPTG induced pGEX-6P-l/M2 containing bacteria lytic products for SDS-PAGE analysis, at the same time, set controls with no-recombinant pGEX-6P-1 bacteria lytic products and sole BL 21 lytic products. The result showed that the recombinant bacteria expressed the expected GST-G2 fusion protein with relative molecular weight of 81000, however, the empty vector of pGEX-6P-1 without inserting the M2 gene only has a specific band at MW 26000.Conclusion1,Plasmid pGEX-6P-1 is a ideal vector for our research on G2 protein of the Hantaan virus;2,pGEX-6P-1/M2 coded GST-G2 fusion protein could be well expressed.3,The fusion protein has biologic activity...