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Hansenula polymorpha mutant and application of expression vector of hansenula polymorpha mutant to recombinant protein expression

A technology of Hansenula and expression vectors, which is applied in the application field of Hansenula mutants and their expression vectors in the expression of recombinant proteins, which can solve the problem of poor gene stability of the host genome, difficulty in screening high-copy strains, and affecting the expression of target proteins Quantity and other issues, to achieve the effect of easy screening, high industrial application value, and short operation time cycle

Active Publication Date: 2015-07-01
JIANGSU THERAVAC BIO PHARMA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although many companies have used this strain for the production of recombinant hepatitis B vaccine, there are still big differences among different manufacturers in the selection of the original strain, and the biomass of the strain is low during fermentation; in addition, the difference in supporting expression vectors leads to host integration. The copy number is not high, it is not easy to screen for high-copy strains, and the stability of the gene integrated into the host genome is poor, which will eventually affect the expression of the target protein and increase the production cost
[0004] The existing yeast-derived recombinant HBsAg is expressed intracellularly, and the purification processes of the manufacturers are different. The mainstream purification process includes steps such as ultracentrifugation, but the process is cumbersome and requires a lot of manpower and material resources. The product The recovery rate is low, which seriously restricts the scale of production

Method used

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  • Hansenula polymorpha mutant and application of expression vector of hansenula polymorpha mutant to recombinant protein expression
  • Hansenula polymorpha mutant and application of expression vector of hansenula polymorpha mutant to recombinant protein expression
  • Hansenula polymorpha mutant and application of expression vector of hansenula polymorpha mutant to recombinant protein expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1. Construction of Hansenula mutant ATCC26012 (ura3-) and determination of its stability and biomass.

[0033] 1. Construction of mutagenic gene fragment 5'UTR-HPURA3Δ40-3'UTR fragment

[0034] Such as figure 1 As shown, the mutagenic gene fragments used for electroporation of Hansenula include URA3 gene upstream UTR 1036bp, downstream UTR 1000bp, and URA3 gene fragments with partial deletions. The construction process is as follows:

[0035] 1. Obtaining the 5'UTR-HPURA3-3'UTR wild-type gene fragment Homo-HPURA3

[0036] Primers were designed according to the reported nucleotide sequence of URA3 of Hansenula (GenBank: X69461.1) as follows:

[0037] Primer 1: 5'-AGAACGTGGACGATAATGACGCAGA-3'

[0038] Primer 2: 5'-GCCGTCTCGATTTGACTACCTCAC-3'

[0039] Use the total genomic DNA of Hansenula Polymorpha ATCC26012 as a template, and use primer 1 and primer 2 to carry out PCR reaction. The specific reaction conditions are: 50 μl reaction system, containing 1 μl KOD-...

Embodiment 2

[0081] Example 2, Construction of uracil auxotrophic Hansenula strain (DL-1.ura3-) supporting expression vector pMOXUR(S.C)KARS1

[0082] 1. Obtain the MOX promoter sequence of methanol oxidase gene by PCR amplification

[0083] Primer 14: 5'- AGATCT TCGACGCGGAGAACGATCTC-3'

[0084] Primer 15: 5'- GAATTC GGTG TGTTGTACTT TAGATT-3'

[0085] The yeast genome extraction kit (Tiangen Biochemical Technology Co., Ltd., Cat. No. DP307-02) was used to extract Hansenula genomic DNA. Using this genomic DNA as a template, use primer 14 and primer 15 to carry out PCR reaction. The underlined parts are the restriction sites of Bgl II and EcoR I respectively. The specific reaction conditions are: 50 μl reaction system, containing 1 μl KOD-Plus polymerase, 5μl 10×Buffer, 5μl dNTP, 2μl 25mM MgSO 4 , 1.5 μl primer 1, 1.5 μl primer 2, 1 μl template, 33 μl ddH2O. The reaction was carried out with a Bio-Rad PCR instrument. The reaction conditions were: 94°C for 2 minutes, cycle once; 94°C ...

Embodiment 3

[0123] Example 3, uracil auxotrophic Hansenula strain (DL-1.ura3-) expressing recombinant hepatitis B surface antigen

[0124] 1. Construction of recombinant expression hepatitis B surface vector

[0125] Hepatitis B surface antigen (HBsAg) amino acid sequence SEQ ID No.1 was retrieved from GenBank. According to the codon preference of Hansenula yeast, it was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. to obtain the sequence: SEQ ID No.2, specifically Sequences can be found in the attached Sequence Listing.

[0126] Primer 32: 5'- GAATTC ATGGAGAACATCACCTCGGG-3'

[0127] Primer 33: 5'- GCGGCCGC TTAGATGTACACCCACAGGCAGA-3'

[0128] Using the Hepatitis B surface antigen (HBsAg) gene sequence synthesized by Jinweizhi as a template, PCR reaction was carried out with primers 32 and 33, and the underlined parts were EcoR I and Not I restriction sites respectively. The specific reaction conditions were: 50 μl reaction system, containing 1 μl KOD-Plus polymerase, 5μl ...

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Abstract

The invention discloses a hansenula polymorpha mutant and application of an expression vector of the hansenula polymorpha mutant to recombinant protein expression, and aims to provide a genetically stable hansenula polymorpha mutant and application of an exclusive expression vector of the genetically stable hansenula polymorpha mutant to recombinant protein expression. The hansenula polymorpha provided by the invention is a mutant stable strain in which the expression of orotidine-5-phosphate decarboxylase gene (UPA3) is blocked. The exclusive expression vector contains a hansenula polymorpha autonomously replicating sequence and a plurality of selection markers, so that the exclusive expression vector can be stably integrated into the strain at a high copy number. The mutant strain and the expression vector can efficiently express a hepatitis B surface antigen, can be correctly assembled into VLP (Virus Like Particles), are simple in process, are very suitable for industrial production of expression proteins, and have great practical application values.

Description

【Technical field】 [0001] The invention relates to the application of a Hansenula mutant and its expression vector in the aspect of recombinant protein expression. 【Background technique】 [0002] There are about 350 million people in the world who are HBV carriers, and 50% to 70% of them are chronic hepatitis B. Studies have shown that about 80% of liver cancer patients are related to hepatitis B. China is a high-incidence area for hepatitis B. There are about 120 million HBV carriers, and about 25% to 40% will eventually die of liver cirrhosis or liver cancer. Currently, there is no curative drug in the world. Due to the problems of carrying HIV and other viruses and limited production, the blood-derived hepatitis B vaccine has been banned by the World Health Organization and replaced by recombinant hepatitis B vaccine. The recombinant hepatitis B vaccine currently circulating in China mainly uses Chinese hamster ovary cells (CHO), Saccharomyces cerevisiae and Hansenula as ...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12P21/02C07K1/36C07K1/34C07K1/18C12R1/85
CPCC07K14/02C12P21/02C12N1/185C12R2001/85
Inventor 李建强任苏林徐晓威葛君周童陈晓晓孙莹孙洪林黄红颖顾月
Owner JIANGSU THERAVAC BIO PHARMA
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