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Housefly cecropin-human lysozyme fusion protein, and preparation method and application thereof

A technology of housefly cecropin and human lysozyme, which is applied in the field of preparation of genetically engineered antibacterial proteins, can solve the problems of not wide antibacterial spectrum, high cost, and inconspicuous antibacterial effect, so as to improve expression efficiency and reduce the probability of tolerance , the effect of low production cost

Inactive Publication Date: 2010-09-01
GUANGDONG PHARMA UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The purpose of the present invention is to provide a new cecropin-human lysozyme with multiple mechanisms of action according to the problems of high cost, non-extensive antibacterial spectrum, and inconspicuous antibacterial effect in the existing peptide antibacterial substances. fusion protein

Method used

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  • Housefly cecropin-human lysozyme fusion protein, and preparation method and application thereof
  • Housefly cecropin-human lysozyme fusion protein, and preparation method and application thereof
  • Housefly cecropin-human lysozyme fusion protein, and preparation method and application thereof

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Embodiment 1

[0035] This program uses the pET expression system of Novagen to express the fusion gene Mdc-hly, the pET32a(+) plasmid is used as the expression vector, and the host cell is Escherichia coli.

[0036] 1 Cloning of the mature peptide sequences of Musca muscae cecropin (Mdc) and human lysozyme (Hly) (see figure 1 )

[0037] According to the instructions of Trizol Reagent, total RNA was extracted from housefly third-instar larvae and human placenta respectively. According to the cDNA sequences of cecropin (Genebank accession number: EF175878) and human lysozyme (Genebank accession number: J03801) published by Genebank, Primer Premier 5.0 Biology software designed 4 specific primers, P1~P4, and the primer sequences are shown in SEQ ID NO:6~9. Primers P1 and P2 amplify the mature peptide sequence of Cecropin, wherein the upstream P1 introduces the Nco I restriction site, the downstream P2 removes the stop codon, adds an asparagine codon AAC and introduces the BamH I restriction sit...

Embodiment 2

[0099] Example 2 Bacteriostatic activity analysis of fusion protein Mdc-hly

[0100] Micrococcus aureus (S.aureus) ATCC25923, M.lysodeikticus (M.lysodeikticus) CICC23645, Bacillus subtilis (B.subtilis) ATCC6633, and Escherichia coli (E. ) ATCC25922, Pseudomonas aeruginosa (P.aeruginosa) ATCC27853, Salmonella (S.paratyphi-B) CICC 21495 MIC value. Dilute Mdc, Hly, and Mdc-hly with sterile Milli-Q water to final concentrations of 200, 100, 75, 50, 25, 12.5, 6.25, 3.121.56, 0.78, and 0.39uM, respectively. Inoculate single colonies into Mueller Hinton (referred to as M-H) medium respectively, after overnight culture, inoculate in fresh M-H medium at a ratio of 1:100, cultivate to logarithmic growth phase at 35°C, count under the microscope on a hemocytometer, and use Adjust the concentration of M-H culture medium to 2×10 6 CFU / ml (colony forming units per ml). Take a sterile 96-well plate, add 100 microliters of freshly prepared bacterial solution to each well, and then add 100 ...

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Abstract

The invention discloses a housefly cecropin-human lysozyme fusion protein, and a preparation method and application thereof. A sequence of the housefly cecropin-human lysozyme fusion protein of the invention is shown by SEQ ID No.1. The housefly cecropin-human lysozyme fusion protein comprises a mature peptide sequence of housefly cecropin, a polypeptide linker sequence and the mature peptide sequence of human lysozyme. The housefly cecropin-human lysozyme fusion protein is prepared by the following steps of: according to the gene sequences of the housefly cecropin and the human lysozyme, designing a synthetic primer with esherichia coli preferred codons; performing PCR amplification by using a splicing overlap extension method to obtain the gene sequence of the fusion protein so as to construct recombinant prokaryotic expression plasmids; transferring the recombinant prokaryotic expression plasmids into host cells to obtain engineering bacteria expressing the fusion protein; and performing fermentation culture, separation and purification to obtain the housefly cecropin-human lysozyme fusion protein of the invention. The fusion protein of the invention has the characteristics of the housefly cecropin and the human lysozyme per se, has relatively more remarkable antibacterial activity and antibiotic activity, can be used for preparing antibacterials or bacteriostatic medicaments, and has wide application prospect.

Description

technical field [0001] The invention relates to the field of preparation of genetically engineered antibacterial proteins, in particular to a fusion protein of housefly cecropin-human lysozyme and its preparation method and application. Background technique [0002] Since the discovery of penicillin, various natural, semi-synthetic and synthetic compounds have been widely and successfully applied to resist various microbial infections. However, with the change of environment and the adaptive evolution of microorganisms, most pathogenic microorganisms are Resistance to traditional antibiotics has become a serious problem affecting public health (Bonomo RA. Multiple antibiotic-resistant bacteria in long-term-care facilities: Anemerging problem in the practice of infectious diseases[J]. Clin Infect Dis, 2000, 31 : 1414-1422.; Murinda SE, Ebner PD, Nguyen LT, et al. Antimicrobial resistance and class 1 integrons in pathogenic Escherichia coli from dairy farms[J]. Foodborne Patho...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K38/47A61P31/00A23L3/3571A23L3/3526A23K1/16A23K1/165A61K38/17
Inventor 朱家勇卢雪梅金小宝马艳梅寒芳曾爱华褚夫江李小波王艳吴强肖明珠
Owner GUANGDONG PHARMA UNIV
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