CN1821398A - Process for preparing heat-labile enterotoxin of E, coli - Google Patents
Process for preparing heat-labile enterotoxin of E, coli Download PDFInfo
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- CN1821398A CN1821398A CNA2006100107324A CN200610010732A CN1821398A CN 1821398 A CN1821398 A CN 1821398A CN A2006100107324 A CNA2006100107324 A CN A2006100107324A CN 200610010732 A CN200610010732 A CN 200610010732A CN 1821398 A CN1821398 A CN 1821398A
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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
The present invention relates to biological technical field, more particularly, the present invention relates to a kind of preparation method who utilizes the yeast cell to express heat-labile enterotoxin of E, coli.
Background technology
Heat-labile enterotoxin of E, coli (heat-labile enterotoxin is called for short LT) is the virulence factor that causes people and animals to suffer from diarrhoea, by enterotoxigenic E (Enterotoxigenic Escherichia, ETEC) synthetic six glycoprotein polyprotein precursors.The 1t full length gene 1148bp of coding and translation heat-labile enterotoxin of E, coli is positioned on the plasmid of wild-type enterotoxigenic E molecular weight about 61 * 10
6U.
LT is made up of two kinds of subunits of A, B, is designated as LTA, LTB respectively, wherein contains the LTB of homology pentamer among the LT.LTA has 258 amino acid, and wherein preceding 18 amino acid are signal peptide; LTB124 amino acid, preceding 21 amino acid are signal peptides.LTB can discern the Ganglioside GM1 of human or animal's intestinal epithelial cell film, and with it in conjunction with form mixture then transmembrane transport enter in the target cell slurry, LTA enters cell, continuous activation adenylate cyclase enzyme system, hydrolysising ATP produces a large amount of cAMP, cause that the intestinal fluid excessive secretion surpasses enteron aisle receptivity again, thereby cause the human or animal to suffer from diarrhoea.
Except very strong toxicity, LT and B subunit thereof also have very strong immunogenicity and can assist a ruler in governing a country other antigens makes body produce specific antibody by mucosal immunity approach, be the potent mucomembranous immunogen and the mucosal adjuvant of generally acknowledging, one of the most effective mucomembranous immunogen of in humans and animals experiment, having identified already so far.LT and B subunit thereof unite with multiple protein antigen or nonprotein antigen or merge behind the common immune body of different immunization routes, but the systemic immunity of enhancing body and mucosal immune response; Simultaneously, can also eliminate body, bring out the permanent immunity memory immunogenic tolerance.Thereby LT holotoxin, especially its nontoxic or low virus mutants have important medical value and economic worth as mucosal adjuvant in mucosal immunity research and in the mucosa immune vaccines exploitation.
Because LT has very high toxicity, thereby hindered the wild-type toxin has been used for human body as the adjuvant of vaccine.The method of transgenation at present commonly used makes wild malicious type LT detoxification keep its adjuvanticity simultaneously, existing surpass 60 kinds of different LT mutant and change its aminoacid sequence by point mutation process and show as low toxicity or nontoxic, fractional mutant wherein mixes with different antigen or the adjuvant experiment has been carried out in fusion on different animal models, and more existing LT mutant are used for clinical trial as the mucosal adjuvant of vaccine.The mutant of common LT A subunit has LTR7K, LTH44A, LTV53D, LTS61F, LTS63K, LTA69G, LTA72R, LTE112K, LTG118E, LTR146E, LTR192G; The mutant of B subunit has LTG33D.
Biology preparation for LT and mutant or subunit, known method is to express LT albumen by genetic engineering means in bacterium, but the output of expressing LT or its mutant or its subunit in bacterium is lower, the LT output of external report all is lower than 20ml/L (Rodighiero et al.J Biol Chem.1999,274 (7): 3962, Uesaka et al.Microb.Pathg, 1994,16 (1): 71; Pronket al.J Biol Chem, 1985,260 (25): 13580; Verwei j et al Vaccine, 1998,16 (20): 2069; Ryan et al.Infect Immun, 1999,67 (4): 1694; Haan et al.Vaccine, 2001,19 (20-22): 2898), domestic king waits secreting, expressing LTB in vibrio aquatilis quietly, and (king waits quietly the highest 26mg/L of being no more than of productive rate, Chinese microbiology and Journal of Immunology, 2003,23 (6): 432); Under the effect of strong promoter, the expression amount in intestinal bacteria is brought up to 46mg/L (Feng Qiang, bioprocess journal, 2003,19 (5): 532 to Feng Qiang etc. with the LT mutant gene; Chinese patent, CN1696291,2004).
The inventor finds that by the gene order of analyzing LT and mutant thereof it is more to contain colibacillary rare codon in the gene order of wild-type LT and mutant thereof, makes that LT and mutant thereof the expression level in intestinal bacteria is lower.But the gene order of wild-type LT and mutant thereof does not have rare codon when using yeast expression, and therefore imagination adopts yeast cell to express wild-type LT and mutant thereof.
In recent years, have with the B subunit of LT respectively pichia spp (Fingerut et al.Vaccine, 2005,23:4685), yeast saccharomyces cerevisiae (Rezaee et al.Journal of microbiology (Seoul, Korea), 2005,43:354; Schonberger et al.Molecular microbiology, 1991, the report of expressing 5:2663), but output is lower, the highest only have 5～8mg/L, all be to have adopted single copy expression cassette, and the yeast cell density OD600 value when producing LTB only has 1.0～2.0, so the output of LTB is lower.In addition, abroad some scholars have also expressed LTB (Wagner etal.J Immunol Methods, 2004,287:203 in plant; Kang et al.Transgenic Res, 2003,12:683; Walmsley et al.Plant Cell Rep, 2003,21:1020; Stop the .S. Mason, Chinese patent CN99814963.2,1999), but productive rate is not high, between 37.8～75 μ g/g.
At present the productive rate of the synthetic wild-type LT of enterotoxigenic E self and the LT that expresses in bacterium, yeast or plant by the genetically engineered mode or its mutant or its subunit is all lower, thereby has limited the large-scale production and the clinical application of LT and mutant thereof.This is the urgency technical barrier to be solved in this area.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, a kind of preparation method of heat-labile enterotoxin of E, coli is provided.This method is a kind of method of expressing LT in yeast cell, and this expression can improve recombination bacillus coli heat labile enterotoxin expression level effectively.
Purpose of the present invention is achieved by following technical proposals.
The invention provides a kind of preparation method of heat-labile enterotoxin of E, coli, this method is that example describes the preparation method with a kind of LT in wild-type LT or its mutant or its subunit and a kind of subunit LTB.
Except as otherwise noted, the percentage ratio that is adopted among the present invention is weight percentage.
Method provided by the present invention, adopt the step of following order:
(1) in yeast cell, carries out expressing in the born of the same parents LT or LTB
The gene of two the subunit A of LT and B is not recombinated on the expression vector pA0815 of yeast cell by pcr amplification and enzyme cutting, form recombinant expression vector LTA/pA0815 and LTB/pA0815, these two recombinant plasmids make up 10 copy heterozygosis recombinant vectorss (AOX-LTB) through the vitro recombination method
10(AOX-LTA)
2The recombinant vectors (AOX-LTB) of/pA0815 or 10 copies
10/ pA0815.Electricity transforms pichia spp after the linearizing of multiple copied recombinant vectors, auxotroph substratum screening yeast transformant, and pcr amplification confirms whether contain goal gene in the transformant.Contain the further liquid fermenting of yeast transformant of goal gene and express with methanol induction.Collect and broken yeast cell, just obtained LT or LTB albumen through affinitive layer purification, SDS-PAG and Western blot identify molecular weight and the immunogenicity of LT and LTB.
(2) secreting, expressing LT and LTB in yeast cell
Recombinate after by pcr amplification and the double digestion downstream of secreting signal peptide alpha factor gene of the gene of the A of LT and two subunits of B, be built into fusion gene α LTA and α LTB, after enzyme is cut, recombinate again on the Yeast expression carrier pA0815, make up multiple copied heterozygosis expression vector (AOX-α LTB) by the vitro recombination method
10(AOX-α LTA)
2/ pA0815 or 10 copy recombinant vectorss (AOX-α LTB)
10/ pA0815, electricity transforms pichia spp after the linearizing of multiple copied recombinant vectors, auxotroph substratum screening yeast transformant, pcr amplification confirms whether contain goal gene in the transformant.Contain the further liquid fermenting of yeast transformant of goal gene and express with methanol induction.Affinitive layer purification has just obtained LT albumen or LTB behind the culture supernatant ultrafiltration and concentration, and SDS-PAG and Western blot identify molecular weight and the immunogenicity of LT and LTB.
(3) evaluation of LT or LTB property of protein
The biologic activity of LT or LTB is to measure the binding ability of itself and Ganglioside GM1 with the ELISA method.The adjuvanticity of LT or LTB adopts LT or LTB and bovine serum albumin (BSA) combined immunization mouse, whether contains secretor type sIgA in the saliva of ELISA detection mouse, the stomach washing fluid, if sIgA is arranged, shows that LT has the mucosal adjuvants activity.
Compared with prior art, the present invention has following beneficial effect:
(1) heterogenous expression LT and mutant thereof or its subunit in the eucaryon yeast cell, can significantly improve its expression level, be because the present invention has made up the LT or the LTB subunit of multiple copied, and expressed two subunits of LT simultaneously, increased LT or the LTB gene dosage in yeast; Secondly, originally LT and mutant thereof in intestinal bacteria rare codon and be not rare codon in yeast cell, can not influence translation and the resultant velocity of LT in yeast cell, finally improve LT and mutant thereof the expression level in yeast cell; At last, the present invention with LT or LTB gene recombination after strong promoter alcoholdehydrogenase (AOX) promotor of yeast expression vector, by methanol induction foreign protein high expression level.
(2) use yeast cell to prepare LT, the contaminated with endotoxins when having avoided traditional method to be prepared by bacterium and the subsequent purification technology that causes loaded down with trivial details makes downstream purification more convenient simple, and clinical use is safer reliable.
(3) if when using the yeast cell secreting, expressing,, reduced purifying process, improved the rate of recovery owing to be not subjected to the influence of host bacterial intracellular protein; Simple in structure, the growth of yeast cell simultaneously fast, be easy to cultivation and fermentation, low, the target protein output height of production cost, this has important practical significance for large-scale production mucosal adjuvants and clinical application thereof.
Description of drawings
Fig. 1 is the multiple copied heterozygosis recombinant plasmid of the interior LT of expression of pichia spp born of the same parents of design construction;
Fig. 2 is the multiple copied heterozygosis recombinant plasmid of the pichia spp secreting, expressing LT of design construction;
Fig. 3 is the multiple copied recombinant plasmid of the interior LTB of expression of pichia spp born of the same parents of design construction;
Fig. 4 is the multiple copied recombinant plasmid of the pichia spp secreting, expressing LTB of design construction;
Fig. 5 is the LTA and the LTB of pcr amplification; (1.100bp DNA ladder Marker; 2.LTA; 3.LTB)
Fig. 6 is the α LTA and α LTB (the 1.DL2000+2700bp Marker of pcr amplification; 2. α LTA; 3. α LTB);
Fig. 7 is 12 copies (AOX-LTB)
10(AOX-LTA)
2The restriction enzyme mapping of/pA0815 (1. λ-EcoT14 I Marker; 2.pA0815 Bgl II and BamH I enzyme cut; 3. (AOX-LTB)
10(AOX-LTA)
2The Bgl II of/pA0815 and BamH I double digestion; 4.DL15000Marker);
Fig. 8 is 12 copies (AOX-α LTB)
10(AOX-α LTA)
2The double digestion collection of illustrative plates of/pA0815 (1. λ-EcoT14 I Marker; 2.DL15000 Marker; 3. (AOX-α LTB)
10(AOX-α LTA)
2The Bgl II of/pA0815 and BamH I double digestion);
Fig. 9 is 10 copies (AOX-LTB)
10The restriction enzyme mapping of/pA0815 (1.pPIC9K/Pst IMarker; 2.DL15000 Marker; 3. (AOX-LTB)
10The Bgl II of/pA0815 and BamH I double digestion);
Figure 10 is 10 copies (AOX-α LTB)
10The restriction enzyme mapping of/pA0815 (1.DL15000 Marker; 2. (AOX-LTB)
10The BamH I enzyme of/pA0815 is cut; 3.AOX-LTB)
10The Bgl II of/pA0815 and BamHI double digestion; 4.pPIC9K/Pst I Marker);
Figure 11 is SDS-PAGE and the Western blot of the LT of expression of recombinant yeast and purifying;
Figure 12 is SDS-PAGE and the Western blot of the LTB of expression of recombinant yeast and purifying.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Because length is limit, in order to express easily, embodiment makes up interior expression of born of the same parents and secreting, expressing wild-type LT or LTB with pichia spp (P.pastoris) and expression vector pA0815 thereof and illustrates.But, it is to be noted, preparation method provided by the present invention also is applicable to other LT mutant fully, for example: LTR7K, LTH44A, LTV53D, LTS61F, LTS63K, LTA69G, LTA72R, LTE112K, LTG118E, LTR146E, LTR192G, LTG33D.
Embodiment 1
---carry out expressing in the born of the same parents LT in the pichia spp cell
(1) structure of recombinant expression vector LTA/pA0815 and LTB/pA0815
LT A and B subunit gene carry out the gene that pcr amplification does not contain its signal peptide sequence with the special primer of its upstream and downstream respectively, and at primer two ends introducing EcoR I enzyme point of contact, cut 2 hours in 37 ℃ of enzymes with EcoR I behind PCR product size LTA (727bp), LTB (320bp) purifying, reclaim purifying; Yeast expression carrier pA0815 cuts with EcoR I enzyme equally, handled 1 hour in 37 ℃ through alkaline phosphatase again, phenol/chloroform extracting, ethanol sedimentation, TE suspending carrier dna fragmentation, be connected in 16 ℃ with the T4 dna ligase respectively with LTB with LTA that EcoR I enzyme is cut and spend the night, connect product and transform by the heat-shocked method and use CaCl
2The E.coli DH5 α of preparation, coating Amp
+-LB flat board was cultivated several single colony inoculation Amp of picking 16 hours for 37 ℃
+-LB nutrient solution, 37 ℃ of shaking culture 12 hours, alkaline lysis extracting plasmid, EcoR I digested plasmid identifies whether contain LTA, LTB, and enzyme is cut the direction of insertion of identifying goal gene then, uses HindIII singly to cut for the LTA/pA0815 recombinant plasmid, stripe size appear after the agarose electrophoresis be 579, during 7847bp, then LTA is that forward inserts, if band is 280, during 8146bp, then be oppositely insertion; Singly cut recombinant plasmid LTB/pA0815 with restriction endonuclease Mun I, occur stripe size behind the electrophoresis and be 119,368,829,1834, show that LTB is that forward inserts during 4877bp,, show it is reverse insertion if occur 108,119,829,2094, the band of 4877bp.Select recombinant plasmid LTA/pA0815, LTB/pA0815 that forward inserts goal gene, be used for next step subclone.
(2) vitro recombination makes up the recombinant plasmid of multiple copy expression cassette
(AOX-LTB)
10(AOX-LTA)
2/pA0815
The LTB/pA0815 recombinant plasmid used 37 ℃ of BglII and BamHI double digestions 2 hours, glue reclaims small segment (AOX-LTB) (1596bp), be inserted into again with BamH I enzyme and cut on the LTB/pA0815 plasmid with alkaline phosphatase treatment, 16 ℃ of connections of T4 dna ligase are spent the night, and transform CaCl with the heat-shocked method
2The E.coli DH5 α competence bacterium of preparation, coating Amp
+-LB flat board was cultivated 16 hours for 37 ℃, picking list bacterium colony, inoculation Amp
+-LB nutrient solution, shaking culture 12 hours, alkaline lysis extracting plasmid, with Bgl II and BamH I double digestion, agarose gel electrophoresis is identified the 2 copy expression cassettes (AOX-LTB) that whether contain 3192bp, 2 (the AOX-LTB)/pA0815 recombinant plasmids that obtain further on its basis, make up recombinant plasmid 5 (the AOX-LTB)/pA0815 that contains 5 copy expression cassettes by above-mentioned same method; Use Bgl II and BamH I double digestion LTA/pA0815 then, reclaiming size is 1995bp (AOX-LTA), and it is inserted among 5 (AOX-LTB)/pA0815 as stated above, forms heterozygosis multiple copy expression cassette recombinant plasmid (AOX-LTB)
5(AOX-LTA)/and pA0815, use Bgl II and BamH I with (AOX-LTB) once more
5(AOX-LTA) two cutting-outs, (AOX-α LTB) recombinates
5(AOX-LTA)/pA0815 on, finally be built into (AOX-LTB)
10(AOX-LTA)
2/ pA0815 heterozygosis multiple copied recombinant plasmid.
(3) (AOX-LTB)
10(AOX-LTA)
2/ pA0815 electricity transforms the evaluation of pichia spp and recombination yeast
(AOX-LTB)
10(AOX-LTA)
2/ pA0815 cut 2 hours in 37 ℃ of enzymes through Sal I, phenol/chloroform extracting, ethanol sedimentation, 20 μ 1ddH
2O suspension DNA, use electroporation under voltage 1500V, resistance 200 Ω, electric capacity 25 μ F, linearization plasmid to be transformed the pichia spp GS115 competence (pressing the pichia spp specification sheets operation of Invitrogen company) of sorbyl alcohol preparation, coating MD flat board, cultivated 3 days for 28 ℃, the bigger bacterium colony of picking a little, with Lyticase enzymic digestion 10 minutes, get 10 μ l and carry out pcr amplification with 5AOX and 3AOX primer, when the appearance size is the band of 913bp, 514bp on the agarose electrophoresis gel, shows and contain LTA and LTB gene in this yeast transformant.So just can filter out the recombination yeast that contains the multiple copy expression cassette goal gene.
(4) abduction delivering, purifying and the evaluation of LT in the pichia spp cell
Picking recombination yeast bacterium colony, be inoculated in the BMGY nutrient solution, (250～300rpm) cultivate in 30 ℃ of vibrations, OD600 reaches at 3～5 o'clock, 3000rpm collected recombinant yeast cell in centrifugal 5 minutes, suspend with the BMMY nutrient solution, continued shaking culture 5 days, centrifugal collection yeast cell, granulated glass sphere grinds smudge cells, TEAN (pH7.3) suspends, 13000rpm, 4 ℃ centrifugal 30 minutes, supernatant liquor is with 0.22 μ m membrane filtration, filtrate is used Immobilized D (+)-galactose affinitive layer purification, the washing purification column, semi-lactosi elution buffer wash-out LT has so just obtained the LT albumen of purifying.Can obtain the purifying LT albumen of 101.3mg by every liter of fermented liquid of above-mentioned experimental arrangement.LT and sample-loading buffer are mixed to be boiled 5 minutes, and get 10 μ l and carry out protein electrophoresis (SDS-PAGE) with 10% polyacrylamide gel, Coomassie brilliant blue dyeing, the molecular weight size of observation LTA and LTB is respectively 27KD and 11KD.LT on the SDS-PAGE is transferred on the pvdf membrane by half-dried electric commentaries on classics method, LT is carried out Immunological Identification according to the schedule of operation of Western blot, the result show LT can with anti-LT antibody generation immune response.
Embodiment 2
---structure and the evaluation of pichia spp secreting, expressing LT
(1) structure of recombinant expression vector α LTA/pA0815 and α LTB/pA0815
LT A and the B subunit gene that does not contain signal peptide sequence carried out pcr amplification respectively, and Xho I and EcoR I enzyme point of contact are introduced in two ends about primer, cut 2 hours in 37 ℃ of enzymes with Xho I and EcoR I behind PCR product LTA (727bp), LTB (328bp) purifying, reclaim purifying; Contain yeast secretary signal peptide α-factor recombinant vectors pALPHA and use Xho I and EcoR I double digestion equally, glue reclaims big fragment, be connected in 16 ℃ with the T4 dna ligase respectively with LTB with the LTA of double digestion and purifying and spend the night, connect product and transform by the heat-shocked method and use CaCl
2The E.coli DH5 α of preparation, coating Amp
+-LB flat board was cultivated several single colony inoculation Amp of picking 16 hours for 37 ℃
+-LB nutrient solution, 37 ℃ of shaking culture 12 hours, alkaline lysis extracting plasmid, Xho I and EcoR I double digestion plasmid identify whether contain LTA, LTB.For the recombinant plasmid that successfully constructs respectively correspondence be designated as p α LTA, p α LTB, LTA and LTB are connected to after α-factor.And then with primer by pcr amplification α LTA, α LTB, all introduce EcoR I restriction enzyme site at the primer two ends, α LTA, α LTB size are respectively 982bp, 583bp.According to the method for (1) among the embodiment 1 α LTA and α LTB are recombinated on the Yeast expression carrier pA0815, be built into α LTA/pA0815, α LTB/pA0815 recombinant vectors.Differentiate for the α LTA direction of insertion on the α LTA/pA0815, use Xho I and EcoR I double digestion, if occur 1058 on the electrophoretic band, then be the forward insertion during 7623bp, occur 584, then be reverse insertion during 8097bp; Use Mun I single endonuclease digestion to identify for the α LTB direction of insertion on the α LTB/pA0815, if occur 119,623,829,1834 on the electrophoretic band, 4874bp then be that forward inserts, if 108,119,829,2349,4877bp then be oppositely insertion.Select recombinant plasmid α LTA/pA0815, α LTB/pA0815 that forward inserts goal gene, be used for next step subclone.
(2) vitro recombination makes up the recombinant plasmid (AOX-α LTB) of multiple copy expression cassette
10(AOX-α LTA)
2/ pA0815
Method according to (2) among the embodiment 1 makes up multiple copied recombinant vectors (AOX-α LTB)
10(AOX-α LTA)
2/ pA0815, difference is that several dna fragmentations vary in size: AOX-α LTA is 2250bp, and AOX-α LTB is 1848bp, and 2 copy expression cassettes (AOX-α LTB) are that 3696,5 (AOX-LTB) are 9240bp, dna fragmentation (AOX-LTB)
5(AOX-α LTB) size is 11490bp, dna fragmentation (AOX-LTB)
10(AOX-α LTB)
2Size is 22980bp.
(3) recombinant plasmid (AOX-α LTB)
10(AOX-α LTA)
2/ pA0815 electricity transforms the evaluation of pichia spp and recombination yeast
According to the method for (3) among the embodiment 1 with (AOX-α LTB)
10(AOX-α LTA)
2/ pA0815 recombinant plasmid electricity transforms pichia spp, carries out pcr amplification pichia spp transformant with 5AOX and 3AOX primer equally, when the appearance size is the band of 1168bp, 769bp on the running gel, shows and contains α LTA and α LTB gene in this yeast transformant.So just can filter out the recombination yeast that contains the multiple copy expression cassette goal gene.
(4) abduction delivering, purifying and the evaluation of pichia spp secretion LT
Picking recombination yeast bacterium colony, be inoculated in the BMGY nutrient solution, (250～300rpm) cultivate, and OD600 reaches at 3～5 o'clock in 30 ℃ of vibrations, 3000rpm collected recombinant yeast cell in centrifugal 5 minutes, suspend with the BMMY nutrient solution, continued shaking culture 5 days, 13000rpm, 4 ℃ are centrifugal 30 minutes, collect culture supernatant, in 4 ℃ of dialysis 3 times, dialyzate is with 0.22 μ m membrane filtration with TEAN (pH7.3) damping fluid, and filtrate is carried out affinitive layer purification and evaluation according to the method for (4) among the embodiment 1.Every liter of fermented liquid of pichia spp secreting, expressing LT can obtain the purifying protein of 126.3mg.
Embodiment 3
---express and secreting, expressing in the born of the same parents of LTB subunit in the pichia spp cell
(1) LTB is in structure, abduction delivering and the evaluation of pichia spp cell inner expression
By the 5 copy expression cassettes (AOX1-LTB) that successfully construct among the embodiment 1
5/ pA0815 recombinant plasmid continues to make up 10 copy expression cassettes (AOX1-LTB) according to the method among the embodiment 1
10/ pA0815 recombinant plasmid, electricity transform pichia spp GS115, identify the pichia spp transformant (fragment with primer 5AOX and 3AOX amplification is 514bp), with methanol induction expression, affinitive layer purification and carry out SDS-PAGE and Western blot evaluation.Size is 11KD on the 10%SDS-PAGE gel, and expressing productive rate in the born of the same parents of LTB is 116.8mg/L.
(2) structure, abduction delivering and the evaluation of LTB secreting, expressing in the pichia spp cell
By the 5 copy expression cassettes (AOX1-α LTB) that successfully construct among the embodiment 2
5/ pA0815 recombinant plasmid continues to make up 10 copy expression cassettes (AOX1-α LTB) according to the method among the embodiment 1
10/ pA0815 recombinant plasmid, electricity transform pichia spp GS115, identify the pichia spp transformant (fragment with primer 5AOX and 3AOX amplification is 769bp), with methanol induction expression, affinitive layer purification and carry out SDS-PAGE and Western blot evaluation.Size is 11KD on the 10%SDS-PAGE gel, and the output of pichia spp secreting, expressing LTB is 143.7mg/L.
Embodiment 4
---biologic activity and the immunological adjuvant activity identification of LT or LTB
The biologic activity of LT or LTB is that its 100 μ l is joined in the elisa plate hole with gm1 gangliosidosis bag quilt, 37 ℃ 1 hour, PBST washes plate three times, adds mouse anti LB antibody 100 μ l, 37 ℃ 1 hour, PBST washes plate three times, the goat anti-mouse antibody that adds 100 μ l HRP marks, 37 ℃ 1 hour, PBST washes plate three times, with 100 μ l O-Phenylene Diamines colour developing liquid colour developing 5 minutes, add 20 μ l 2NH
2SO
4Color development stopping is that 492nm measures the OD value with the microplate reader wavelength.The result shows that LT and LTB have and gm1 gangliosidosis bonded ability.
LT or LTB and antigen bovine serum albumin (BSA) are by Al (OH)
3After the colloid absorption,, collect the saliva of mouse and the washing fluid and the serum of casing slime by the oral time immune mouse 0,2,4 Wednesdays that carries out.Can detect the antibody that contains anti-BSA in the serum by the ELISA test kit, and contain the sIgA antibody of anti-BSA in saliva and the casing slime, show that the LT of yeast cell to express or LTB have the mucosal adjuvants activity.
Claims (3)
1. the preparation method of a heat-labile enterotoxin of E, coli, this method adopts the step of following order:
(1) in yeast cell, carries out expressing in the born of the same parents LT or LTB
The gene of two the subunit A of LT and B is not recombinated on the expression vector pAO815 of yeast cell by pcr amplification and enzyme cutting, form recombinant expression vector LTA/pAO815 and LTB/pAO815, these two recombinant plasmids make up the recombinant expression vector that the heterozygosis recombinant expression vector or 10 that comprises 10 copy LTB and 2 copy LTA copies LTB through the vitro recombination method; Electricity transforms pichia spp after the linearizing of multiple copied recombinant expression vector, auxotroph substratum screening yeast transformant, and pcr amplification confirms whether contain goal gene in the transformant; Contain the further liquid fermenting of yeast transformant of goal gene and express with methanol induction; Collect and broken yeast cell, just obtained LT or LTB albumen through affinitive layer purification, SDS-PAG and Western blot identify molecular weight and the immunogenicity of LT and LTB;
(2) secreting, expressing LT or LTB in yeast cell
Recombinate after by pcr amplification and the double digestion downstream of secreting signal peptide alpha factor gene of the gene of the A of LT and two subunits of B, be built into fusion gene α LTA and α LTB, after cutting, enzyme recombinates again on the Yeast expression carrier pAO815, make up on pAO815 by the vitro recombination method and to contain 10 copy α LTB and 2 copy α LTA heterozygosis recombinant expression vectors or 10 copy α LTB recombinant expression vectors, electricity transforms pichia spp after the linearizing of multiple copied recombinant vectors, auxotroph substratum screening yeast transformant, pcr amplification confirms whether contain goal gene in the transformant; Contain the further liquid fermenting of yeast transformant of goal gene and express with methanol induction; Affinitive layer purification has promptly obtained LT or LTB albumen behind the culture supernatant ultrafiltration and concentration, and SDS-PAG and Western blot identify molecular weight and the immunogenicity of LT and LTB.
2. the preparation method of heat-labile enterotoxin of E, coli according to claim 1, wherein said yeast cell is a Pichia.
3. the preparation method of heat-labile enterotoxin of E, coli according to claim 1, wherein said heat-labile enterotoxin of E, coli has comprised wild-type, various mutant LTR7K, LTH44A, LTV53D, LTS61F, LTS63K, LTA69G, LTA72R, LTE112K, LTG118E, LTR146E, LTR192G, LTG33D and its B subunit.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103773790A (en) * | 2012-10-24 | 2014-05-07 | 财团法人台湾动物科技研究所 | Mutant escherichia coli heat-repellent toxin, preparation method thereof, adjuvant for increasing immune response and vaccine |
| CN104031935A (en) * | 2014-05-23 | 2014-09-10 | 宁波大学 | Intracellular expression method of large yellow croaker liver expression antibacterial peptide LEAP-2C in pichia pastoris |
| CN109467606A (en) * | 2018-11-15 | 2019-03-15 | 大连理工大学 | A kind of escherichia coli enterotoxin STa-LTB-STb fusion protein and its encoding gene and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2002533068A (en) * | 1998-12-22 | 2002-10-08 | ボイス トンプソン インスティテュート フォア プラント リサーチ | Orally immunogenic bacterial enterotoxin expressed in transgenic plants |
| CN1696291A (en) * | 2004-07-15 | 2005-11-16 | 中国人民解放军第三军医大学 | New type mutant of heatlabile enterotoxin from bacteria coli, and preparation method |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103773790A (en) * | 2012-10-24 | 2014-05-07 | 财团法人台湾动物科技研究所 | Mutant escherichia coli heat-repellent toxin, preparation method thereof, adjuvant for increasing immune response and vaccine |
| CN104031935A (en) * | 2014-05-23 | 2014-09-10 | 宁波大学 | Intracellular expression method of large yellow croaker liver expression antibacterial peptide LEAP-2C in pichia pastoris |
| CN109467606A (en) * | 2018-11-15 | 2019-03-15 | 大连理工大学 | A kind of escherichia coli enterotoxin STa-LTB-STb fusion protein and its encoding gene and application |
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