Method for expressing pseudoplectania nigrella mature peptide in recombinant pichia pastoris

A mature polypeptide, Pichia pastoris technology, applied in the field of genetic engineering, can solve the problems of plasmid instability, few people use, easy to lose, etc.

Active Publication Date: 2010-09-29
刘德虎
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] At present, the expression of exogenous proteins in Pichia pastoris is generally through the integration of exogenous genes into the Pichia genome, although there are also some expression plasmids (such as 2μ) carrying foreign genes tha

Method used

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  • Method for expressing pseudoplectania nigrella mature peptide in recombinant pichia pastoris
  • Method for expressing pseudoplectania nigrella mature peptide in recombinant pichia pastoris
  • Method for expressing pseudoplectania nigrella mature peptide in recombinant pichia pastoris

Examples

Experimental program
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Effect test

Embodiment 1

[0049] The artificial synthesis of the mature polypeptide gene of embodiment 1 pseudosigladenin

[0050] According to the known full-length gene sequence (GenBank: AJ964941) of Pseudomycin, according to the codons preferred by Pichia pastoris, without changing the amino acid sequence, artificially synthesized the gene encoding the mature polypeptide of Pseudomycin , the synthetic mature polypeptide gene has a full length of 123bp (including the stop codon), encodes a total of 40 amino acid residues (see SEQ ID NO: 1), and has a molecular weight of about 4.4KDa. During the process of artificially synthesizing the polypeptide nucleotide coding sequence, a restriction enzyme site XhoI and a yeast Kex2 gene expression product cleavage recognition sequence were synthesized and added before the first codon GGT at the 5' end of the gene CTCGAGAAAAAGA (see SEQ ID NO: 2); a restriction enzyme cutting site Not I was added after the stop codon TAA at the 3' end of the gene. 19 nucleoti...

Embodiment 2

[0051] The cloning of embodiment 2 pseudosigladenin mature polypeptide gene

[0052] The above-mentioned artificially synthesized pseudosigalcin mature polypeptide gene fragment was directly inserted into the T site of the pEASY-T1 (purchased from Beijing TransGenic Company) plasmid to obtain a bacterial clone containing the plasmid vector mp-T, and then, through DNA Sequencing to determine the correctness and completeness of the mature pseudosigladin gene contained in it (DNA sequencing was completed by Beijing Biaokai Technology Co., Ltd.).

Embodiment 3

[0053] Example 3 Cloning of Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase promoter

[0054] According to the known Pichia glyceraldehyde-3-phosphate dehydrogenase promoter sequence (GenBank: U62648.1), synthesize primers F and R located at both ends of the promoter, wherein F is 5'-AT GGATCC TTTTTTGTAGAAATGTCTTGGTGTCC-3' (the underline is the BamH I cleavage site), R is 5'-AT GAGCTC TGTGTTTTGATAGTTGTTCAATTGATTG-3' (the underline is the Sac I cleavage site), using Pichia pastoris genomic DNA as a template (for the genome extraction method, see "Molecular Cloning Experiment Guide, Third Edition"), using F and R as primers, amplified by PCR. The glyceraldehyde-3-phosphate dehydrogenase promoter sequence (GAPDH, whose sequence is shown in SEQID NO: 3) was obtained by increasing, and the amplified fragment was directly inserted into the T site of the pEASY-T1 plasmid to obtain an intermediate plasmid containing Bacterial clones vectoring GAPDH-T were then analyzed by ...

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Abstract

The invention provides a method for expressing and producing pseudoplectania nigrella mature peptide recombinant proteins in pichia pastoris cells in a constitutive type mode, which comprises the following steps of: 1) optimizing pseudoplectania nigrella mature peptide genes; 2) obtaining pichia pastoris glyceraldehyde-3-phosphate dehydrogenase promoter DNA sequences by adopting methods such as molecular cloning and the like, and placing the pseudoplectania nigrella mature peptide genes under the regulation and control of the glyceraldehyde-3-phosphate dehydrogenase promoters to connect with plasmids to obtain new, constitutive and secretory type pichia pastoris efficient expression vectors; 3) converting the pichia pastoris by using the vectors and obtaining the recombinant pichia pastoris strains to obtain the pseudoplectania nigrella mature peptide with bioactivity; and 4) optimizing the high-density fermentation conditions of the engineering strains and the expression and purification of the recombinant protein. The pseudoplectania nigrella mature peptide prepared by the method can be applied in the fields of medicinal treatment, food, feed, scientific research and the like and is suitable for the scale production of pseudoplectania nigrella protein.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for expressing a mature pseudosantagactin polypeptide in recombinant Pichia pastoris. Background technique [0002] Antimicrobial peptides (AMPs) is a general term for a class of small molecule polypeptides composed of 12-100 amino acids, with a net positive charge, amphipathic and antibacterial activity. The research on antimicrobial peptides has a history of more than 50 years. So far, about 880 antimicrobial peptides from various biological sources have been identified (Brogden K A, 2005). Antimicrobial peptides have attracted increasing attention due to their broad-spectrum antibacterial activity (including against Gram-positive bacteria, Gram-negative bacteria, fungi, and even some viruses, etc.). Especially in the past few decades, due to the abuse of antibiotics, pathogenic strains with antibiotic resistance have emerged continuously, and there is an urgent nee...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/31C12N1/19C07K14/37C07K1/16C12R1/84
Inventor 刘德虎
Owner 刘德虎
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