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Glpks3 and its coding gene related to the synthesis of Pneumonia Kangding b0

A kind of technology of Feisangkangding and gene, which is applied in the field of the synthesis related protein glpks3 and its coding gene of Feisangkangding B0, which can solve the problem of high price and achieve the effect of increasing production

Active Publication Date: 2016-03-23
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no domestically produced caspofungin product, which must be imported for domestic use, and the price of a single tube is as high as 1,000 yuan

Method used

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  • Glpks3 and its coding gene related to the synthesis of Pneumonia Kangding b0
  • Glpks3 and its coding gene related to the synthesis of Pneumonia Kangding b0
  • Glpks3 and its coding gene related to the synthesis of Pneumonia Kangding b0

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1, discovery of glpks3 protein and its coding gene

[0054] The whole genome of Glarealozoyensis was sequenced. After assembly, gene prediction and annotation of the sequencing results, a polyketide synthase was found, and the protein was named glpks3 protein.

[0055] The glpks3 protein is shown as sequence 1 in the sequence listing. The gene encoding glpks3 protein is named glpks3 gene, the genomic DNA is shown in sequence 2 of the sequence listing, and the open reading frame in the cDNA is shown in sequence 3 of the sequence listing.

Embodiment 2

[0056] Example 2, functional verification of glpks3 protein and its coding gene

[0057] 1. Construction of glpks3 gene knockout vector pAg1-H3-glpks3

[0058] 1. According to the sequence of glpks3 gene, design two pairs of primers as follows:

[0059] PrimerC and PrimerD constitute primer pair A, and the target sequence is the 473rd to 3522nd nucleotides from the 5' end of sequence 2 in the sequence listing.

[0060] Primer C: 5'-CC TTAATTAA GGACTTCTTCTAGAGGTTG-3' (underlined PacI recognition sequence);

[0061] Primer D: 5'-G ACTAGT CTGTCACTTCGTTTGCG-3' (SpeI recognition sequence is underlined).

[0062] PrimerA and PrimerB constitute primer pair B, and the target sequence is the 4332nd to 8512th nucleotides from the 5' end of sequence 2 in the sequence listing.

[0063] Primer A: 5'-ca GGGCCC TCAGCAAGACTACTTCTC-3' (the ApaI recognition sequence is underlined);

[0064] Primer B: 5'-ca CCCGGG TTAACAACGACTTGTGAG-3' (XmaI recognition sequence is underlined).

[0...

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Abstract

The invention discloses a method for synthesizing associated protein glpks3 through pneumocandin B0 and an encoding gene of the glpks3. Protein provided in the method can be (a) protein composed of an amino acid sequence presented in a sequence 1 in a sequence table, or (b) protein associated with pneumocandin B0 synthesis, derived from the sequence 1 and formed in the mode that the amino acid sequence presented in the sequence 1 in the sequence table undergoes substitution and / or deletion and / or adding through one or more amino acid residues. By means of expression of the encoding gene for prohibiting the protein in Glarea lozoyensis, the yield of the pneumocandin B0 is improved by four times. Theoretical bases are provided for intensive study of metabolic regulation of pneumocandin biosynthesis, the method can be used for improving the yield of the pneumocandin B0 through gene knockout, and guarantee is provided for high yield of caspofungin.

Description

technical field [0001] The present invention relates to a kind of lung capsule Kangding B 0 Synthesis of related protein glpks3 and its coding gene. Background technique [0002] Caspofungin (Caspofungin) is the first Echinocandin (Echinocandin) antifungal drug approved by the US FDA, which can specifically inhibit the growth of fungal cell wall β-(1,3)-D-glucan. Synthetic, dissolves fungi, has good antifungal activity, and does not produce cytotoxicity to patients like amphotericin B and other drugs, so it is also known as penicillin in antifungal drugs. [0003] Caspofungin is clinically used to treat patients with invasive aspergillosis and candidemia bacteremia who are ineffective or intolerable to other treatments and secondary candidal abdominal abscess and peritonitis. In 2009, its global sales amounted to US$617 million, accounting for 12.45% of the total antifungal drug hospital market in China, ranking fourth. At present, there is no domestically produced caspof...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/04C12N15/80C12N1/15C12N9/10C12N15/54C12N5/10C12R1/645
Inventor 刘杏忠安志强岳群陈里向梅春
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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