Study On The Strategy Of Preparing HBV CccDNA In Vitro

Part 1: The strategy of constructing HBV cccDNA in vitro Objective: This study aimed to develop a novel tool to study the function of cccDNA in clinical HBV drug-resistant genotypes by exploring a convenient and rapid method to synthesize HBV cccDNA in vitro,whose functionality was verified at the cell level.Method: According to the special structure of HBV rcDNA,PCR primers with negative strand as template and positive strand as template were designed to carry out two PCR amplification of HBV genome,and the two PCR products were used to construct cccDNA in vitro based on the seam Less cloning technology of Gibson Assembly after modifying the premix formulation of this method.The HBV cccDNA product constructed by this method was extracted and purified by phenol chloroform,and then transfected into liver cancer cell lines such as Huh7 and Hep G2.After 5-7days,the cells and the culture medium were separated,then virus core particle DNA were extracted,and the number of copies of the virus replication was determined by real-time quantitative PCR;HBeAg and HBsAg in the supernatant of the culture medium were determined by ELISA method.This method is applied to clinical drug-resistant samples for subsequent functional analysis and research by constructing circular DNA of drug-resistant genotypes.Result: The method of constructing circular HBV cccDNA in vitro was successfully established.HBV cccDNA was constructed in vitro from two truncated HBV DNA fragments by the improved Gibson seam Less cloning method.The cells after transfection can secrete HBeAg,HBsAg and other corresponding HBV molecular markers,which shows cccDNA constructed in vitro has biological function.Compared with the reported one-step method of amplifying HBV full-length DNA followed by enzyme digestion,self-ligation and circularization and then transfection,this method is faster and more convenient;the cost of improved self-made Gibson reaction solution is lower than commercial reagents.Conclusion: The method of constructing HBV cccDNA in vitro has been successfully established,and it is promising to be used in clinical genotyping analysis of large quantities of drug-resistant samples.Part 2:The protein of SARS-CoV-2 is expressed in vitro and identifiedObjective: The prokaryotic expression plasmid is constructed with SARSCoV-2 RBD gene inserted,and the recombined plasmid is expressed in Escherichia coli by IPTG-induction to obtain functional recombinant RBD proteins.The eukaryotic expression plasmid is constructed with SARSCoV-2 Nucleocapsid gene inserted,and the recombined plasmid is expressed in GS115 Pichia yeast by methanol-induction to obtain functional recombinant N proteins.Method:(1)The DNA sequence of the RBD region of SARS-CoV-2 spike protein is analyzed,and the rare codons are optimized by using bioinformatics software.The optimized sequence is synthesized in vitro and inserted into prokaryotic expression vector.The RBD recombinant plasmid is transformed into Rosetta competent cells to induce the expression of exogenous proteins.The recombinant RBD protein is identified and then combined with human angiotensin-converting enzyme 2(ACE2)in vitro to verify its function.(2)The DNA sequence of SARSCoV-2 Nucleocapsid protein is synthesized in vitro and inserted into eukaryotic expression vector.The N recombinant plasmid is transformed into GS115 competent cells to induce the secretory expression of exogenous proteins.The recombinant N protein is identified by SDSPAGE and Western Blot.Result:(1)Some specific proteins are expressed in broken bacteria supernatant of pGEX6P-1/RBD,which are identified as soluble recombinant GST-RBD proteins.The recombinant GST-RBD protein can bind to human ACE2 protein in vitro with strong affinity.HIS-RBD protein mostly exists in the form of inclusion bodies in pET28a/RBD.After purification and renaturation,it can specifically bind to ACE2 with strong affinity up to 129 nmol by ITC;N protein was successfully secreted and expressed in GS115,and a large amount of N protein could be detected in the medium supernatant.Conclusion: IPTG-induction enable to be success of soluble recombinant RBD protein expression in Escherichia coli.The recombinant proteins specifically bind to human ACE2 receptor and can be used in many aspects such as SARS-CoV-2 antiviral drug research and vaccine development.