Process for preparing heat-labile enterotoxin of E, coli

A heat-labile, Escherichia coli technology, applied in the biological field, can solve the problems of limiting the large-scale production and clinical application of LT and its mutants, low LTB yield, low yield, etc., to avoid endotoxin contamination and low production costs. , the effect of improving the recovery rate

Inactive Publication Date: 2006-08-23
KUNMING UNIV OF SCI & TECH
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AI Technical Summary

Problems solved by technology

[0008] In recent years, the B subunit of LT has been separated in Pichia pastoris (Fingerut et al. Vaccine, 2005, 23: 4685), Saccharomyces cerevisiae (Rezaee et al. ; Schonberger et al. Molecular microbiology, 1991, 5: 2663), but the yield is low, the highest is only 5-8 mg / L, all of which adopt a single-copy expression cassette and the yeast cell density when producing LTB The OD600 value is only 1.0-2.0, so the yield of LTB is low
In addition, some foreign scholars also expressed LTB in plants (Wagner et al. J Immunol Methods, 2004, 287: 203; Kang et al. Transgenic Res, 2003, 12: 683; Walmsley et al. : 1020; Hugh. S. Mason, Chinese patent CN99814963.2, 1999), but the yield is not high, between 37.8~75 μg / g
[0009] At present, the yields of wild-type LT synthesized by toxigenic Escherichia coli and LT or its mutants or subunits expressed in bacteria, yeast or plants through genetic engineering are low, which limits the scale of LT and its mutants Chemical production and clinical application

Method used

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  • Process for preparing heat-labile enterotoxin of E, coli
  • Process for preparing heat-labile enterotoxin of E, coli
  • Process for preparing heat-labile enterotoxin of E, coli

Examples

Experimental program
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Embodiment 1

[0039] - Intracellular expression of LT in Pichia pastoris cells

[0040] (1) Construction of recombinant expression vectors LTA / pA0815 and LTB / pA0815

[0041] The LT A and B subunit genes use their upstream and downstream specific primers to amplify the gene without its signal peptide sequence by PCR respectively, and introduce EcoR I enzyme cutting points at both ends of the primers. The PCR product sizes are LTA (727bp), LTB ( 320bp) after purification, digested with EcoR I at 37°C for 2 hours, recovered and purified; yeast expression vector pA0815 was also digested with EcoR I, then treated with alkaline phosphatase at 37°C for 1 hour, extracted with phenol / chloroform, and precipitated with ethanol , TE suspension carrier DNA fragments, and EcoR I digested LTA and LTB were ligated with T4 DNA ligase at 16°C overnight, and the ligated products were transformed by heat shock method with CaCl 2 Prepared E.coli DH5α, coated with Amp + -LB plate, cultured at 37°C for 16 hours...

Embodiment 2

[0050] ——Construction and identification of LT secreted and expressed by Pichia pastoris

[0051] (1) Construction of recombinant expression vectors αLTA / pA0815 and αLTB / pA0815

[0052]The LTA and B subunit genes without signal peptide sequences were amplified by PCR respectively, and Xho I and EcoR I restriction sites were introduced at the upper and lower ends of the primers, and the PCR products LTA (727bp) and LTB (328bp) were purified and used Xho I and EcoR I were digested at 37°C for 2 hours, recovered and purified; the recombinant vector pALPHA containing the yeast secretion signal peptide α-factor was also double digested with Xho I and EcoR I, the large fragment was recovered from the gel, and the double digested and purified LTA and LTB were ligated overnight at 16°C with T4 DNA ligase, and the ligated products were transformed by heat shock method with CaCl 2 Prepared E.coli DH5α, coated with Amp + -LB plate, cultured at 37°C for 16 hours, picked several single c...

Embodiment 3

[0060] ——Intracellular expression and secretory expression of LTB subunits in Pichia pastoris cells

[0061] (1) Construction, induced expression and identification of LTB expressed in Pichia pastoris cells

[0062] Construct successful 5 copy expression cassettes (AOX1-LTB) in embodiment 1 5 / pA0815 recombinant plasmid, continue to construct 10 copies of expression cassette (AOX1-LTB) according to the method in embodiment 1 10 / pA0815 recombinant plasmid, electrotransformation of Pichia pastoris GS115, identification of Pichia pastoris transformants (the fragment amplified with primers 5AOX and 3AOX is 514bp), induction of expression with methanol, purification by affinity chromatography, and SDS-PAGE and Western blot Identification. The size on 10% SDS-PAGE gel is 11KD, and the intracellular expression yield of LTB is 116.8 mg / L.

[0063] (2) Construction, induced expression and identification of LTB secreted and expressed in Pichia pastoris cells

[0064] The successful...

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Abstract

The present invention discloses the preparation process of heat labile enterotoxin of E. coli, and the preparation process has intracellular expression or secretory expression of LT or LTB in yeast cell. Exogenously expressing LT and its mutant or subunit in eukaryotic yeast cell has greatly raised expression level. The present invention constitutes multicopy LT or LTB subunit expression kit, has increased gene dosage of LT or LTB in yeast, LT or LTB gene recombination following alcohol dehydrogenase promoter as the powerful promoter of Pichia yeast expression vector, methanol induced high expression of exogenous protein in yeast cell and thus raised expression level of LT and its mutant or LTB in yeast cell, simple post purification and safe clinical application. The present invention has low production cost and high target protein yield, and is significant in the scale production and clinical application of mucous membrane adjuvant.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically, the invention relates to a preparation method for expressing Escherichia coli heat-labile enterotoxin by using yeast cells. Background technique [0002] Escherichia coli heat-labile enterotoxin (LT for short) is a virulence factor that causes diarrhea in humans and animals. It is a hexameric protein synthesized by toxigenic Escherichia coli (Enterotoxigenic Escherichia, ETEC). The 1t gene encoding and translating Escherichia coli heat-labile enterotoxin is 1148 bp in full length, located on the plasmid of wild-type toxigenic Escherichia coli, with a molecular weight of about 61×10 6 u. [0003] LT is composed of two subunits, A and B, which are denoted as LTA and LTB respectively, and LT contains homopentamer LTB. LTA has 258 amino acids, of which the first 18 amino acids are signal peptides; LTB has 124 amino acids, and the first 21 amino acids are s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N1/19C12N15/81C12N15/31C12P21/02C12R1/84
Inventor 井申荣魏云林林连兵李光
Owner KUNMING UNIV OF SCI & TECH
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