Hepatitis B treatment vaccine base on inactivated whole recombinant Hansenula polymorpha cells which expresses HBsAg and HBcAg

A hepatitis B therapeutic vaccine comprising an inactivated whole recombinant Hansenula polymorpha cells, comprising and expressing an HBsAg comprising an amino acid sequence of SEQ ID NO:

• 2 encoded by a nucleic acid sequence of SEQ ID NO:
• 1 and comprising and expressing an HBcAg comprising an amino acid sequence of SEQ ID NO:
• 4 encoded by a nucleic acid sequence of SEQ ID NO:
• 3, wherein the recombinant Hansenula polymorpha cells comprises 15-21 HBcAg-specific CTL epitopes, the HBsAg is adw subtype, the intracellular expression level of the HBsAg in the recombinant Hansenula polymorpha cells is 6-10 μg HBsAg per 108 cells, the HBsAg has 16-21 HBsAg-specific CTL epitopes, and wherein the inactivated whole recombinant Hansenula polymorpha cells are an adjuvant, wherein a construction method of an engineering strain of the HBsAg expressed by the recombinant Hansenula polymorpha cells includes the following steps:
• (1) transforming a plasmid having the DNA sequence as shown in SEQ ID NO:
• 1 into an URA3-auxotrophic Hansenula cell strain HU-11 (CGMCC No. 1218) of a host cell by cell electroporation, and picking up single colony transformants grown on a MD selection culture plate;
• (2) selecting colonies with fast growth rate, using semi-quantitative PCR to detect the brightness of HBsAg gene band, selecting colonies with high copy numbers, and screening for 20 to 400 generations in successive subcultures;
• (3) screening multiple copies of heterologous integrated transformed clones after the successive subcultures in step (2), inducting with methanol for 72 hours, and determining the expression level of HBsAg after disruption of the transformed cells by radioimmunoassay;
• (4) high-copy, high-expression clones with free plasmids being screened and removed through step (3), and HBsAg gene copy number being detected by quantitative PCR;
• and
• (5) basing on the test results of step (4), 30 liters of fermented genetically stabilized recombinant Hansenula HBsAg strain is selected as a primary strain of the engineering strain.