Construction of a eukaryotic Hansenula engineering bacterium containing recombinant hepatitis B virus gene and production method of hepatitis B surface antigen
A technology of Hansenula and engineering bacteria, applied in the fields of genetic engineering, virus/bacteriophage, biochemical equipment and methods, etc.
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Embodiment 1
[0044] Example 1: Cloning of Hansenula methanol oxidase promoter and terminator
[0045] According to the known sequences (GenBank: A11156, AR363832 and E00783), primers MOX_P-F and MOX_P-R located at both ends of the MOX gene promoter were synthesized, respectively,
[0046] Wherein MOX_P-F is 5'-CCAATAGATCTTCGACGCGGAGAACGATCTCCTC-3',
[0047] MOX_P-R1 is 5'-GGAACCTCCACCAACAACAATGATATCGAAT-3', and the primers MOX_TT-F1 and MOX_TT-R1 located at both ends of the MOX gene terminator are synthesized,
[0048] Where MOX_TT-F1 is 5'-TCGGAACTTACGAGGAGACCGGACTTGCCAG-3',
[0049] MOX_TT-R1 is 5′-CTTGTGTCTCACACCCATAATGATCCCGTT-3′, using Hansenula genomic DNA as a template (for the genome extraction method, see page 485 of "Molecular Cloning Experiment Guide, Third Edition"), the MOX promoter and terminator were amplified by PCR , the amplified fragment was directly inserted into the pEASY-Blunt-Zero plasmid, and according to the method provided by the company, the bacterial clone con...
Embodiment 2
[0050] Embodiment 2: Construction of expression vector pHPZF1.0
[0051] The MOX-P promoter was amplified by PCR from the pMOX-P plasmid, and cloned into the vector pPICZC using the BglII-EcoRI site to obtain the plasmid pPICZC-MOXP; according to the sequence of the MOX promoter obtained by sequencing verification, the primer MOX_P-R2 was redesigned. With MOX_P-F and MOX_P-R2 as primers,
[0052] Where MOX_P-F is 5'-CCAATAGATCTTCGACGCGGAGAACGATCTCCTC-3' (the underlined part is the BglII site), MOX_P-R2 is 5'-CACGTGAATTCCTCGTTTCGAAGCTTTGTTTTTGTACTTTAGATT-3' (the underlined part is the EcoRI site), and the intermediate plasmid vector pMOX-P DNA was used as a template (see page 485 of "Molecular Cloning Experiment Guide, Third Edition" for the plasmid extraction method), and the MOX promoter was amplified by PCR, and BglII and EcoRI sites were introduced at both ends of the PCR product. Double digestion was performed with restriction endonuclease BglII-EcoRI, and the DNA fragmen...
Embodiment 3
[0056] Example 3: Analysis of the consensus amino acid sequence of adr subtype HBsAg
[0057] Since genotype C includes all adr serotypes, we used the standard genome sequence AY123041 of hepatitis B virus type C reported in Japan as the retrieval sequence, and Blast NCBI nucleic acid database (similarity parameters were set to 93-100%) to retrieve the HBV genome sequence Nearly 2000 entries. After investigation, 617 HBV genome sequences of genotype C reported in China were sequenced. Excluding sequencing errors and HBsAg nonsense mutation sequences, a total of 479 HBsAg protein sequences of adr serotype reported in China were obtained (25 of which were from Hong Kong sequences and 4 of them were from Hong Kong). from Taiwan). Use the function of BioEdit software to perform amino acid sequence comparison analysis, and obtain the most representative HBsAg consensus amino acid sequence (consensus amino acid sequence, that is, the sequence of amino acid residues with the highest...
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