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Method for producing HPV52 L1 protein by using Hansenula polymorpha expression system

By using the dual screening strategy of Zeocin and G418 in the Hansenula yeast system and increasing the induction temperature, combined with the use of POROS XS chromatography medium, the efficient expression and purification of HPV52 L1 protein was successfully achieved, solving the problem of HPV52 L1 protein expression in the existing technology. and difficult purification problems, providing an effective method for vaccine preparation.

Active Publication Date: 2019-11-22
BEIJING ABZYMO BIOSCIENCES CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In terms of the induced expression of foreign proteins, the induction time of HPV16 and HPV18 L1 in the fermenter needs to be more than 20 hours, and no specific information on the protein expression level is provided
In addition, as an important indicator of protein purification, there is no disclosure about the purification process and the purity of the exogenous proteins HPV16 and HPV18 L1 when the purification is completed

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Analysis of HPV52 L1 consensus amino acid sequence

[0056] The full-length HPV52 L1 protein is composed of 503 amino acids. After searching in GenBank, the AlignX function of Vector NTI software was used for amino acid sequence comparison and analysis, and the most representative HPV52 L1 consensus amino acid sequence (consensus amino acid sequence) was obtained. Each amino acid position of L1 adopts the sequence of the most frequently occurring amino acid residue, and its sequence is shown in SEQ ID NO:1.

Embodiment 2

[0057] Example 2: Optimized design and artificial synthesis of HPV52 L1 coding gene

[0058] In order to use Hansenula to efficiently express HPV52 L1 protein, the inventors performed codon optimization of the nucleotide sequence for Hansenula according to the amino acid sequence shown in SEQ ID NO:1. The optimization principles include: a) Use the most frequently used or higher codons according to the Hansenula genetic code frequency table; b) Avoid negative regulatory elements that have potential effects on gene transcription or protein translation, such as PolyAT region, PolyGC region, Sliencer region and internal splicing sites, etc.; c) Comprehensive analysis of mRNA secondary structure including 5'UTR, HPV52L1 coding region and 3'UTR, to avoid the formation of complex RNA secondary structure, Reduce the free energy of mRNA secondary structure; d) Use a 5'UTR region that is completely consistent with the natural sequence downstream of Hansenula promoter as far as possible up...

Embodiment 3

[0060] Example 3: Generation of expression constructs carrying HPV52L1 nucleotide sequence

[0061] The Hansenula expression vector used in the present invention is the Hansenula expression vector pRMHP2.1 (SEQ ID NO: 9) described in the Chinese patent application with application number 201210021524.X.

[0062] (1) PCR amplification of MOX promoter and MOX terminator

[0063] Using the mixed genomic DNA of Hansenula strains ATCC26012 and ATCC34438 as templates, the following primer pairs were used to amplify the MOX promoter with a size of 1518 bp, and the NotI restriction site was introduced upstream;

[0064] Upstream primer of MOX promoter: 5'-AAGGAAAAAAGCGGCCGCAACGATCTCCTCGAGCTGCTCGC-3' (SEQID NO: 3)

[0065] Downstream primer of MOX promoter: 5'-TTTGTTTTTGTACTTTAGATTGATGTC-3' (SEQ ID NO: 4)

[0066] Using the mixed genomic DNA of Hansenula strains ATCC26012 and ATCC34438 as templates, the following primer pairs were used to amplify the MOX terminator with a size of 311bp, and at th...

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Abstract

The invention relates to a method for producing HPV52 L1 protein by using a Hansenula polymorpha expression system, specifically to a method for producing recombinant Hansenula polymorpha cells for expressing the HPV52 L1 protein, and recombinant Hansenula polymorpha cells produced by the method. The invention also discloses a method for producing the HPV52 L1 protein by using the recombinant Hansenula polymorpha cells and application of the produced HPV52 L1 protein in the preparation of a preventive vaccine.

Description

[0001] This application is a divisional application of the invention patent application with the filing date of April 26, 2013, the application number being 201310150032.5, and the invention title being "Method for Producing HPV52 L1 Protein with Hansenula Expression System". [0002] Invention field [0003] The invention belongs to the technical field of medical bioengineering, and relates to a method for producing HPV52 L1 protein, in particular to a method for producing HPV52 L1 protein using a Hansenula expression system. Background technique [0004] Human papillomavirus (HPV) is a closed-loop double-stranded DNA virus without envelope. It belongs to the subfamily Papillomavirus and Polyomaviruses. It mainly invades the epithelial mucosal tissue of the human body and induces various benign and malignant Hyperplastic lesions. [0005] At present, more than 200 different subtypes of HPV have been identified. HPV infection has obvious tissue specificity. Different types of HPV have...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19C12N15/37C07K14/025A61K39/12A61P31/20C12R1/78
Owner BEIJING ABZYMO BIOSCIENCES CO LTD
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