Recombinant DNA sequence, hansenula polymorpha, preparation method for hepatitis B surface antigen, and hepatitis B vaccine
A technology of DNA sequence and surface antigen, which is applied in the field of genetic engineering to achieve high yield, high genetic stability and high expression level
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[0040] The present invention also provides a method for preparing the surface antigen of the adr subtype hepatitis B virus, which comprises: fermenting and culturing the recombinant yeast as described in any one of the above items, so as to obtain the surface antigen of the adr subtype hepatitis B virus.
[0041] Preferably, the method further includes: after fermenting and culturing the recombinant yeast, performing cell disruption, filtration, adsorption, and hydrophobic chromatography, so as to prepare further purified adr subtype hepatitis B virus surface antigen.
[0042] Preferably, the filtration is ultrafiltration; the adsorption is silica gel adsorption; and the hydrophobic chromatography is butyl agarose hydrophobic chromatography.
[0043] The present invention also provides a hepatitis B vaccine, characterized in that the vaccine comprises the surface antigen of the adr subtype hepatitis B virus obtained by any one of the above-mentioned preparation methods.
[004...
Embodiment 1
[0046] Obtaining of embodiment 1 recombinant DNA sequence
[0047] According to the scientific literature "A.K.Raney and A.McLachlan, The Biology of Hepatitis B Virus, In: Editor A.Mclachlan ed. Molecular Biology of the Hepatitis B Virus, 2000, CRC Press." Obtain the adr subtype hepatitis B virus surface antigen amino acid sequence. The above nucleic acid sequence encoding HBsAg was redesigned with reference to the codon frequency of the Hansenula gene (see US Patent No. 5,712,114).
[0048] During the design, according to the amino acid sequence, the Hansenula optimal coding for the adr subtype hepatitis B virus surface antigen was artificially obtained from the codon frequency of the Hansenula gene (see US patent document US5712114), and the restriction endonuclease site was searched with DNAMAN software point, excluding BamHI and EcoRI restriction sites.
[0049] According to the above design, a sequence composed of 678 bases was obtained, as shown in SEQ ID NO: 1 in the ...
Embodiment 2
[0050] Example 2 Construction of recombinant Hansenula polymorpha
[0051] The pMPT-02 plasmid (obtained from Tianjin Bohui Biotechnology Co., Ltd.) was digested with EcoRI and BamHI (the total volume of the reaction system: 50 μl, including 5 μl of the B buffer system of Roche Reagent Company, EcoRI 1.5 μl; BamHI 1.5 μl, pMPT- 02 plasmid 10 μl, temperature is 37°C, reaction time is 1 hour), electrophoresis to recover the digested vector fragment, use the DNA recovery kit of Treasure Bio (Dalian) Co., Ltd. and recover the digested vector fragment according to its instructions) According to the steps described in the appendix of TaKaRa PCR Ligation Kit (Code No.D6022, obtained from Treasure Bioengineering (Dalian) Co., Ltd.), the vector fragment was connected to the insert fragment shown in SEQ ID NO: 3 described in Example 1 Afterwards, thermal transformation was carried out, and the thermal transformation conditions were as follows: 5 μl of the ligated recombinant plasmid was...
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