Recombinant polymorphic hansenula polymorpha and preparation method thereof

A technology of Hansenula polymorpha and yeast, applied in the field of bioengineering, can solve the problems of low reactivity, difficulty in inoculating hepatitis B vaccine, escape mutation, etc., to reduce the probability of contamination, good immune protection effect, and mutation sites clear effect

Active Publication Date: 2013-05-08
BEIJING BIOLOGICAL PROD INST CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, existing vaccines still have their shortcomings: 5%-10% of the population has no response or low response to this type of vaccine even if the dose is increased; escape mutations may occur after vaccination; and the vaccine itself is relatively large. The high dosage and high price make it practically difficult to universally vaccinate hepatitis B vaccine in poor developing countries
[0006] The current HBV vaccine production uses S protein as the only immunogen, and the antigenic composition is relatively simple

Method used

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  • Recombinant polymorphic hansenula polymorpha and preparation method thereof
  • Recombinant polymorphic hansenula polymorpha and preparation method thereof
  • Recombinant polymorphic hansenula polymorpha and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] The synthesis of the gene sequence of S, M, L protein in the embodiment 1HBV

[0092] The S gene, M gene and L gene sequences of the adr subtype of HBV were searched from GenBank, and were optimized according to the codon usage preference of Hansenula polymorpha before sequence synthesis, and the following sequences were respectively obtained: SEQ ID No. 7 (S protein), SEQ ID No.8 (M protein), and SEQ ID No.9 (L protein), for specific sequences, please refer to the attached sequence listing. Whole gene synthesis was performed on the above-mentioned sequences of SEQ ID No.7, SEQ ID No.8, and SEQ ID No.9.

Embodiment 2

[0093] The amplification of embodiment 2 promoter, terminator

[0094] Using Hansenula (ATCC26012) genome as a template, use HSDNA polymerase (TakaRa, Code No.DR010S) PCR amplifies the promoters and terminators of methanol oxidase gene (MOX), dihydroxyacetone synthase gene (DAS), methanol dehydrogenase gene (FMD).

[0095] Primers were designed according to the GenBank MOX promoter (MOX-P, see SEQ ID No.1) and terminator (MOX-T, see SEQ ID No.2) nucleotide sequence:

[0096] MOX(P)-F: C GAGCTC AATTGTTGCCGCATGCATCCTTGC (SacI)

[0097] MOX(P)-B: TTTGTTTTTGTACTTTTAGATTGATGT

[0098] MOX(T)-F: GGAGACGTGGAAGGACATACCGC

[0099] MOX(T)-B: CGG GTC GAC ACAAAAGCTGGAGCTCAATCTCCGG (Sal I);

[0100] According to GenBank DAS promoter (DAS-P, see SEQ ID No.3) and terminator (DAS-T, see SEQ ID No.4) nucleotide sequence primers are:

[0101] DAS(P)-F: C GAGCTC AAGCTTGAATCGTGAAACGTCG (SacI)

[0102] DAS(P)-B: GGGAAGAAAAGACAGAGATG

[0103] DAS(T)-F: CCACGATAAAGTAAATAAGC

[0104] D...

Embodiment 3

[0122] Example 3 Obtaining of Exogenous Gene Fragment Expression Cassette

[0123] 1. Integrated arm connection

[0124] Using primers 25S-F1, 25S-S1 and 25S-F2, 25S-B2 to amplify the upper and lower gene sequences 25S1 (SEQ ID No.10), 25S2 (SEQ ID No.11) of the 25S gene in the Hansenula genome, respectively , as the integration arm of homologous recombination between yeast expression vector and yeast chromosome, connected into pUC-57, and the specific process of forming pUC-25S is as follows:

[0125] 25S-F1: CCG GAATTC TCGCTTCTTCACATTC (EcroI)

[0126] 25S-B1: C GAGCTC GGGATATGGATTTAG (SacI)

[0127] 25S-F2: ACGC GTC GAC AATCTAAATTATTG (SalI)

[0128] 25S-B2: CCC AAGCTT CGAGCTTTTCAGATAATTGG (HindIII)

[0129] The PCR reaction system for amplifying the 25S1 target gene sequence is shown in Table 2 below:

[0130] Table 2 The PCR reaction system used to amplify the 25S1 target gene sequence

[0131] Template: yeast genomic DNA

2μL

25S-F1

1...

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Abstract

The invention provides a recombinant polymorphic hansenula polymorpha and a preparation method thereof. The recombinant polymorphic hansenula polymorpha comprises an encoding gene containing L protein of hepatitis B virus, and the encoding gene containing S protein of the hepatitis B virus. Compared with the prior art, the polymorphic hansenula polymorpha provided by the invention has the following advantages and positive effects that the polymorphic hansenula polymorpha can be independently configured into VLP (Virus Like Particle) in a human body, and the VLP is closest to HBV (Hepatitis B Virus) natural virus particles, so that the immune protective effect of the VLP to the HBV infection is theoretically higher. The polymorphic hansenula polymorpha a provided by the invention is excellent in hereditary stability, adopts methanol or glycerin as the only and one carbon source, realizes the repression/derepression inducible expression mode and enables extrinsic proteins to be efficiently expressed.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a recombinant Hansenula polymorpha and a preparation method thereof. Background technique [0002] Hepatitis B virus (HBV) infection is a very serious global health problem. There are about 350 million HBV chronically infected people in the world, about 75% are distributed in the Asia-Pacific region, many of them develop chronic liver diseases such as liver cirrhosis, primary liver cancer, etc., and about 1 million people die from hepatitis B virus every year in the world Infection-related diseases. At present, there is no specific drug for the treatment of hepatitis B, and the most important means of controlling the spread of hepatitis B is vaccination with effective vaccines. Commercial hepatitis B vaccine has been put into clinical use for more than fifteen years, and hepatitis B vaccine has been included in the WHO's Expanded Immunization Program (EPI). [0003] T...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12R1/78
Inventor 李启明张靖张学峰梁宇靳玉琴马智静陈实于洁
Owner BEIJING BIOLOGICAL PROD INST CO LTD
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