CN100537771C - A kind of colibacillus periplasm solinocrine type expression vector - Google Patents
A kind of colibacillus periplasm solinocrine type expression vector Download PDFInfo
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- CN100537771C CN100537771C CNB2003101170374A CN200310117037A CN100537771C CN 100537771 C CN100537771 C CN 100537771C CN B2003101170374 A CNB2003101170374 A CN B2003101170374A CN 200310117037 A CN200310117037 A CN 200310117037A CN 100537771 C CN100537771 C CN 100537771C
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Images
Abstract
The present invention relates to a kind of colibacillus periplasm solinocrine type expression vector pFUW802s, this carrier is by adding polygalacturonase (PelB) signal peptide (SEQ ID NO:1) at the N of foreign protein end, and make foreign protein and FK506 binding protein peptide acyl prolyl cis-trans isomerase (FkpA) coexpression, improve foreign protein expression level in the colibacillus periplasm chamber; A kind of pericentral siphon solinocrine type is expressed the carrier pFUW802s/CEA-scFv of anticancer embryonal antigen single-chain antibody (scFv); A kind of carrier pFUW802s/hSLC of pericentral siphon solinocrine type expressing human secondary lymphoid-tissue chemokines; And a kind of host cell that contains above-mentioned pericentral siphon solinocrine type expression vector.
Description
Technical field
The present invention relates to a kind of colibacillus periplasm solinocrine type expression vector, more specifically, relate to a kind of by adding PelB signal peptide (MKYLLPTAAAGLLLLAAQPAM at the N of foreign protein end, (SEQ ID NO:1)), and make foreign protein and FK506 binding protein peptide acyl prolyl cis-trans isomerase coexpression, improve the new expression vector of the expression level of foreign protein in the colibacillus periplasm chamber; The construction process of this expression vector; And the host cell that contains this kind carrier.
Technical background
Compare with eukaryotic expression system (as yeast, insect cell, mammalian cell etc.), prokaryotic expression system (mainly being coli expression system) has the advantage of many uniquenesses., cell clear such as genetic background growth rapidly, fermentation period is short, be easy to screening and gene recombination operation, expression system is than horn of plenty etc.But also there are some shortcomings in prokaryotic expression system: can't glycosylation and be easy to form inclusion body etc. as foreign protein.The inclusion body that foreign protein forms must pass through sex change, fold renaturation again, just can obtain active protein product.The said process operation is loaded down with trivial details, annealing efficiency is low, need be optimized respectively for different foreign proteins.Therefore, if can adopt prokaryotic expression system to realize soluble-expression or the expression of pericentral siphon solinocrine in the born of the same parents of foreign protein, will increase work efficiency greatly in application in practice so, (Swartz JR.et al., 2001) save production cost.
People taked multiple mode to promote the soluble-expression of foreign protein.With SP (SPA) (Abrahmsen L.et al., 1986; Uhlen M.et al., 1989), gsh (GST) (Smith DB.et al., 1988; Sharrocks AD.et al., 1994) or sulphur oxygen cyclase protein (Thioredoxin) (LaVallie ER.Et al., 2003) amalgamation and expression, can play short solvable effect to the part foreign protein; Utilize protein disulfide to form associated protein (Kurokawa Y.et al., 2000,2001; Zhang Z.et al., 2002), cis-trans propyl isomerism enzyme (Zhang Z.et al., 2003) and other molecular chaperoneses (Humphereys DP.et al., 1996; ) folding assisted effect, the design expression vector can obviously promote soluble-expression in the born of the same parents of foreign protein.But the soluble-expression mode also has apparent in view shortcoming in the born of the same parents: belong to the reductibility environment in the born of the same parents, be unfavorable for that foreign protein forms correct disulfide linkage bridge, and then influence the formation (Ritz D.et al., 2001) of correct structure picture; And need ultrasonication or pressure breaking cell walls could discharge soluble-expression component in the born of the same parents, be unfavorable for large scale culturing and purifying.
Comparatively speaking, because the pericentral siphon chamber belongs to oxidized form environment (Ritz D.et al., 2001), help forming correct disulfide linkage bridge, and extract and not need fracturing cell walls when component is expressed in the pericentral siphon chamber, can reduce the purifying cost significantly, be a kind of ideal phraseology so pericentral siphon solinocrine type is expressed.Usually foreign protein and appropriate signals peptide are merged, foreign protein is secreted in the pericentral siphon chamber by the guiding function of signal peptide.The copy number of control expression vector, select suitable promotor and substratum, adopt suitable abduction delivering mode and culture condition (culture temperature, induce opportunity and induction time), can improve efficient (the Mergulhao FJ.et al. that pericentral siphon solinocrine type is expressed, 2003a, 2003b).
The folding process of protein in the colibacillus periplasm chamber exists three main late incident (Missiakas D.et al.1997): the formation of disulfide linkage, the isomerization of proline(Pro).These incidents form associated protein (as DsbA, B, C, D, E, G etc.), prolyl cis-trans isomerase (as FkpA etc.) catalysis by protein disulfide respectively and finish, and its efficient has directly determined the efficient that the pericentral siphon solinocrine type of exogenous protein is expressed.Have and experiment showed, that the foreign protein that is secreted in the pericentral siphon chamber also forms the inclusion body of non-activity sometimes.The molecular chaperones that accessory protein in the pericentral siphon chamber is folding is incorporated in the secreted expression carrier, not only helps improving pericentral siphon solinocrine type expression level, but also can promote the correct folding of foreign protein, improves the activity of expression product.
The inventor's colleague is on the basis of PET28a+ carrier (Novagen), respectively with Dsbc DsbG (Zhang Z.et al., 2002) or FkpA (Zhang Z.et al., 2002) with foreign protein amalgamation and expression and coexpression, obviously improve the ratio of soluble-expression in the born of the same parents, but do not found the raising of pericentral siphon solinocrine type expression level.So when using pericentral siphon chamber molecular chaperones, need to promote the method combination that pericentral siphon solinocrine type is expressed with other, could realize efficient pericentral siphon solinocrine type expression.DsbA and insulin precurosor (pro-insulin) amalgamation and expression have been realized the latter's secretion type expression (WinterJ.et al., 2001).But the foreign protein of amalgamation and expression need be removed the non-target protein part that merges through proteolytic cleavage, has increased operation steps and production cost, reduces production efficiency, is unfavorable for the mass preparation target protein.
Summary of the invention
Summary of the invention
The present invention creatively is incorporated into FkpA in the structure of intestinal bacteria secreted expression carrier, and adopts the mode of coexpression, a series of foreign proteins that add signal peptide has successfully been carried out pericentral siphon solinocrine type expressed.
Therefore, one aspect of the present invention provides a kind of and is incorporated into the general pericentral siphon solinocrine type expression vector of intestinal bacteria that makes up on the coli expression carrier with gene engineering method by urging folding associated protein FkpA.
In another aspect of the present invention, provide be useful on the host cell that makes up the general pericentral siphon solinocrine type expression vector of intestinal bacteria a kind of containing.
In another aspect of the present invention, provide a kind of intestinal bacteria general pericentral siphon solinocrine type expression vector pFUW802s.
In another aspect of the present invention, provide a kind of expression vector pFUW802s/CEAscFv that pericentral siphon solinocrine type is expressed anti-anticancer embryonal antigen (CEA:Carcino Embryonic Antign) single-chain antibody that is used for.
In another aspect of the present invention, provide a kind of carrier pFUW802s/hSLC that is used for pericentral siphon solinocrine type expressing human secondary lymphoid-tissue chemokines (hSLC:Human Secondary Lymphoid-tissue Chemokine).
In another aspect of the present invention, a kind of host cell that contains general pericentral siphon solinocrine type expression vector pFUW802s is provided, described host cell is preferably intestinal bacteria.
In another aspect of the present invention, the host cell of a kind of pFUW802s/CEAscFv1 of containing or pFUW802s/hSLC carrier is provided, described host cell is preferably intestinal bacteria.
In another aspect of the present invention, provide a kind of method that makes up above-mentioned various carriers.
In another aspect of the present invention, a kind of method of the formal representation foreign protein with pericentral siphon solinocrine type is provided, it is characterized in that utilizing above-mentioned general pericentral siphon solinocrine type expression vector
At the context of this specification sheets, especially on the basis of the disclosure of " embodiment " part, other aspects and advantages of the present invention are conspicuous to those skilled in the art.
Description of drawings
Fig. 1 is the building process sketch of the general pericentral siphon solinocrine type expression vector pFUW802s of intestinal bacteria.
Fig. 2 is used to make up the intermediate carrier pFUW802 of the general pericentral siphon solinocrine type expression vector pFUW802s of intestinal bacteria and the sketch of pTCEF.
Fig. 3 is the synoptic diagram of the general pericentral siphon solinocrine type expression vector pFUW802s of intestinal bacteria.
Fig. 4 utilizes the general pericentral siphon solinocrine type expression vector of the constructed intestinal bacteria of the present invention SDS-PAGE figure and Western blot figure with the anti-CEA single-chain antibody of pericentral siphon solinocrine type formal representation in intestinal bacteria.
A figure is SDS-PAGE result, swimming lane 1: the molecular weight of albumen standard; Swimming lane 2:pFUW802 pericentral siphon chamber component (P component); The ultrasound precipitation component of swimming lane 3:pFUW802 (UD component); The ultrasonic supernatant component (US component) of swimming lane 4:pFUW802; The P component of swimming lane 5:pFUW802/CEAscFv; The UD component of swimming lane 6:pFUW802/CEAscFv; The US component of swimming lane 7:pFUW802/CEAscFv; The P component of swimming lane 8:pFUW802s/CEAscFv; The UD component of swimming lane 9:pFUW802s/CEAscFv; The US component of swimming lane 10:pFUW802s/CEAscFv; The P component of swimming lane 11:pFUW802s; The UD component of swimming lane 12:pFUW802s; The US component of swimming lane 13:pFUW802s.Thick arrow indication is FkpA, and thin arrow indication is anti-CEA single-chain antibody.
B figure is Western blot result, swimming lane 1: the molecular weight of albumen standard; The P component of swimming lane 2:pFUW802s/CEAscFv; The UD component of swimming lane 3:pFUW802s/CEAscFv; The US component of swimming lane 4:pFUW802s/CEAscFv; Swimming lane 5:pFUW802/CEAscFvP component; The UD component of swimming lane 6:pFUW802/CEAscFv; The US component of swimming lane 7:pFUW802/CEAscFv.
Fig. 5 utilizes the general pericentral siphon solinocrine type expression vector of the constructed intestinal bacteria of the present invention activation analysis result (antigen-binding specificity of the anti-CEA single-chain antibody of elisa assay) with the anti-CEA single-chain antibody of pericentral siphon solinocrine type formal representation in intestinal bacteria.
Fig. 6 utilizes the general pericentral siphon solinocrine type expression vector of the constructed intestinal bacteria of the present invention SDS-PAGE figure and Western Blot figure with pericentral siphon solinocrine type formal representation people's secondary lymphoid-tissue chemokines (hSLC) in intestinal bacteria.
The SDS-PAGE of pericentral siphon solinocrine type formal representation hSLC and Western blot identify.A figure is SDS-PAGE result, the P component of swimming lane 1:pFUW802/hSLC; Swimming lane 2: molecular weight of albumen standard.Swimming lane 3-5: the P component of different clones' pFUW802s/hSLC.B figure is Western blot result, swimming lane 1: the molecular weight of albumen standard; The P component of swimming lane 2:pFUW802/hSLC; The P component of swimming lane 3:pFUW802s/hSLC.
Detailed Description Of The Invention
In the context of the present invention, employed term generally has ability unless otherwise indicated The implication that the those of ordinary skill in territory is understood usually. Especially, following term has following implication:
" short folding GAP-associated protein GAP " as herein described refers to folding and ripe mistake any and protein The protein that journey is associated, comprise various molecular chaperone proteins, protein disulfide form albumen, Protein disulfide cis-trans isomerase, protein peptidyl prolyl cistrans isomerase etc.
The short folding GAP-associated protein GAP FkpA that the present invention adopts has peptide acyl proline cis-trans isomerism simultaneously The activity of enzymatic activity and molecular chaperones (Ramm K.et al., 2000,2001; Bothmann H.et al., 2000; ArieJP.et al., 2001). FkpA albumen is the albumen of Escherichia coli constitutive expression, its Ripe body is arranged in the colibacillus periplasm chamber, mainly plays a role at this position. But when the external source egg During white great expression, script obviously can not satisfy crowd like this with the amount of the FkpA that foundation level is expressed The folding requirement of many foreign proteins. By the encoding gene of FkpA is cloned into multicopy shape Formula is present on the plasmid in the Escherichia coli, so that FkpA also can great expression, thus effectively slow Separated the deficiency of short folding GAP-associated protein GAP, a large amount of foreign proteins is correctly folded. To urge folding GAP-associated protein GAP FkpA and be incorporated on the colibacillus periplasm solinocrine type expression vector, logical Cross in pericentral siphon chamber overexpression this short folding GAP-associated protein GAP and just can effectively improve foreign protein Expression promotes folding effect with playing.
" coexpression " as herein described refers to exogenous gene sequence and short folding related protein gene order Row can be transcribed under the control of same transcripting promoter, but turn over respectively with different translation units Be translated into short folding GAP-associated protein GAP and external source destination protein; Exogenous gene sequence and short folding GAP-associated protein GAP Gene order also can be transcribed respectively under the control of different transcripting promoters, and with different Translation unit is translated into respectively short folding GAP-associated protein GAP and external source destination protein.
In a word, carrier provided by the present invention has following characteristics:
1. the short folding GAP-associated protein GAP FkpA of coexpression effectively promotes foreign protein in the pericentral siphon chamber Correct folding, improve pericentral siphon solinocrine type expression;
2. when adopting this carrier to carry out the expression of pericentral siphon solinocrine type, do not need broken bacteria cell wall, Directly extract pericentral siphon chamber component and namely can gather in the crops destination protein.
3. the albumen of expressing by this way all is the activated protein that function is arranged, and does not need step numerous Trivial and denature and renature process that efficient is extremely low only needs comparatively simple purification step just can obtain A large amount of foreign proteins;
4. this expression vector is take Escherichia coli as expressive host, but bulk fermentation production is produced Technology is simple, and operation is with low cost easily.
Embodiment
Below in conjunction with specific embodiment, and the present invention is described in further detail with reference to accompanying drawing.Should be understood that the embodiment in this specification sheets is in order to demonstrate the invention, but not limit the scope of the invention by any way.
Embodiment:
Laboratory, inventor place has successfully made up difunctional expression vector (phage display and expression foreign protein) pFUW80 (Zhang Weiguo etc., Acta Genetica Sinica, 1999,26 (2) .99-106) and pFUW802.The present invention adopts gene engineering method reconstruction pFUW802, makes it to have new multiple clone site; Introduce short folding associated protein FkpA simultaneously, enable to carry out coexpression with foreign protein under the control of same transcripting promoter, the final structure finished the general pericentral siphon solinocrine of intestinal bacteria type expression vector pFUW802s.Adopt the enzyme blanking method will resist CEA single-chain antibody, people's Lymphoid tissue chemokine to be connected in the above-mentioned carrier, and transformed into escherichia coli HB2151, carry out pericentral siphon solinocrine type and express.Adopt the method for salt shock to extract pericentral siphon chamber protein ingredient, carry out SDS-PAGE and Western Blot and identify, antagonism CEA single-chain antibody expression product adopts ELISA methods analyst antigen-binding activity.
The structure of embodiment 1. pericentral siphon solinocrine type expression vector pFUW802s (make up route and see Fig. 1)
1. change the multiple clone site of difunctional expression vector pFUW802.
According to the building process of Fig. 1, at first design a pair of primer (primer 1:5 '-GGCCGCGAATTCAAATTCTATTTCGGATCCGGC-3 ' (SEQ ID NO:2); Primer 2: 5 '-GGCCGCCGGATCCGAAATAGAATTTGAATTCGC-3 ' (SEQ ID NO:3)), forms one section double chain DNA fragment after both anneal, all form restriction enzyme site NotI automatically at these segmental two ends.Concrete grammar is as follows: each 1 μ l of primer 1 and primer 2, add water 8 μ l, and on the common PCR instrument, carry out 10 following reactions repeatedly: 94 ℃ of 30sec, 72 ℃ of 30sec.Reaction product is used for following ligation directly as the external source fragment.
Adopt plasmid that the Shen, Shanghai can betting office to extract test kit in a small amount and extract carrier pFUW802 (concrete operations reference reagent box specification sheets) about 1 μ g, carry out NotI (the precious biotech company in Dalian) single endonuclease digestion reaction then, 37 ℃ of 2 hours.Reaction mixture is formed: the carrier pFUW802 about 1 μ g (10 μ l), 2 μ l, 10 * damping fluid, 1 μ l NotI, 7 μ l distilled waters.Enzyme is cut product after 1% agarose electrophoresis, adopts the glue recovery test kit of Shen, Shanghai energy betting office to reclaim big fragment.Reclaim fragment and be dissolved in the 50 μ l distilled waters, be used for following ligation.
The ligation mixture: 2 μ l external source fragments, 15 μ l enzymes are cut product, 1 μ l T4DNA ligase enzyme (the precious biotech company in Dalian), 2 μ l, 10 * T4 dna ligase.16 ℃ connect 2hours.Connect product and be used for transformed into escherichia coli TOP10 (Novagen).
According to " molecular cloning experiment guide " (Jin Dongyan, Li Mengfeng translates, 1996, Science Press) the 2nd edition method prepares the competent cell of intestinal bacteria TOP10.Getting 1 μ l connection product joins in the 100 μ l competent cells, after placing 1hr on ice, 42 ℃ of heat shock 90sec place 2min then on ice, and (10g/l tryptone is (available from GIBCO company to add 800 μ l LB substratum, down together), 5g/l yeast extract (available from GIBCO company, down together), 5g/l NaCl, pH7.5), hatch 1hr for 37 ℃.Get wherein 200 μ l be coated with the LB-A flat board (10g/l tryptone, 5g/l yeast extract, 5g/l NaCl, 15g/l agar and 100 μ g/ml penbritins, pH7.5), 37 ℃ of overnight incubation.
Picking mono-clonal colony inoculation LB-A substratum (10g/l tryptone, 5g/l yeast extract, 5g/l NaCl, 100 μ g/ml penbritins, pH7.5), 37 ℃ of shaking tables are cultivated 12hr, adopt the plasmid of Shen, Shanghai energy betting office to extract test kit extraction plasmid in a small amount, and PCR identifies whether the external source fragment is correctly inserted.PCR method: with general upstream primer (5 ' aaattctatttcaaggagacagtcata3 ' (SEQ ID NO:4)) and the pairing of aforementioned primer 2 of pFUW802, increase as primer, can expand the positive clone of the clone who the special band about 200bp, called after pFUW802r1.PCR reaction conditions: in the amplification system of 20 μ l, add 0.1～1 μ l plasmid DNA (about 20～200ng) and each 10pmol of upstream/downstream primer, the dNTPs of 2 μ l, 10 * Taq enzyme buffer liquid and 2 μ l 2mM, Taq enzyme (self-control) 1U, moisturizing is to cumulative volume 20 μ l.Behind 94 ℃ of pre-sex change 5min, 94 ℃ of sex change 40Sec, annealing 40Sec, 72 ℃ are extended 40Sec, 25 circulations of increasing, last sample 5 μ l adopt 1% agarose gel electrophoresis to detect.Through said process, polyclone point among the pFUW802 and fd phage gene 3 (fd gene 3) are replaced by one section nucleotide sequence (SEQ ID NO:5) that contains a series of restriction enzyme sites and replace.This sequence and appended restriction enzyme site are as follows.
2. make up pericentral siphon solinocrine type expression vector pFUW802s
Adopt plasmid that the Shen, Shanghai can betting office to extract test kit in a small amount and extract carrier pTCEF and pFUW802r1 about 1 μ g, carry out endonuclease reaction then respectively.Reaction conditions is as follows: 37 ℃ of enzymes are cut 2hr.Reaction mixture is formed: the carrier about 1 μ g (10 μ l), 2 μ l, 10 * damping fluid, 1 μ l EcoRI and 1 μ l BamHI, 6 μ l distilled waters.Enzyme is cut product after 1% agarose electrophoresis, adopts the glue recovery test kit of Shen, Shanghai energy betting office to reclaim.The pTCEF enzyme is cut small segment product (containing ribosome bind site: RBS and FkpA gene order) and pFUW802r1 enzyme and is cut and all be dissolved in the 50 μ l distilled waters after big fragment product reclaims, and is used for following ligation.
The ligation mixture: 6 μ l pFUW802r1 enzymes are cut big fragment product, and 20 μ l pTCEF enzymes are cut the small segment product, 1 μ l T4 dna ligase (the precious biotech company in Dalian), 3 μ l, 10 * T4 dna ligase.16 ℃ connect 2hours.Connect product and be used for transformed into escherichia coli TOP10.Method for transformation is the same.37 ℃ of shaking tables of picking mono-clonal colony inoculation LB-A substratum are cultivated 12hr, adopt the plasmid of Shen, Shanghai energy betting office to extract test kit extraction plasmid in a small amount, because it is big than the obvious change of pUW802r1 to be connected into after the FkpA gene order size of carrier, adopts 1% agarose electrophoresis just can identify positive colony.The positive colony carrier is pericentral siphon solinocrine type expression vector pFUW802r2.
Adopt plasmid that the Shen, Shanghai can betting office to extract test kit in a small amount and extract carrier pFUW802 and pFUW802r2 about 1 μ g, carry out endonuclease reaction then respectively.Reaction conditions is as follows: 37 ℃ of enzymes are cut 2hr.Reaction mixture is formed: the carrier about 1 μ g (10 μ l), 2 μ l, 10 * damping fluid, 1 μ l SacII and 1 μ l EcoRI, 6 μ l distilled waters.Enzyme is cut product after 1% agarose electrophoresis, adopts the glue recovery test kit of Shen, Shanghai energy betting office to reclaim.The pFUW802 enzyme is cut small segment product (about 300bp contains Plac, ribosome bind site RBS and PelB signal peptide gene sequence) and pFUW802r2 enzyme and is cut and all be dissolved in the 50 μ l distilled waters after big fragment product reclaims, and is used for following ligation.
The ligation mixture: 6 μ l pUW802r2 enzymes are cut big fragment product, and 20 μ l pFUW802 enzymes are cut the small segment product, 1 μ l T4 dna ligase (the precious biotech company in Dalian), 3 μ l, 10 * T4 dna ligase.16 ℃ connect 2hours.Connect product and be used for transformed into escherichia coli TOP10.Method for transformation is the same.37 ℃ of shaking tables of picking mono-clonal colony inoculation LB-A substratum are cultivated 12hr, adopt the plasmid of Shen, Shanghai energy betting office to extract test kit extraction plasmid in a small amount, and PCR identifies whether the external source fragment is correctly inserted.PCR method: with the general upstream primer of pFUW802 (5 ' aaattctatttcaaggagacagtcata 3 ' (SEQ ID NO:4)) and the pairing of aforementioned primer 2, increase as primer, can expand the positive clone of the clone who the special band about 300bp, called after pFUW802s.PCR reaction conditions: in the amplification system of 20 μ l, add 0.1～1 μ l plasmid DNA (about 20～200ng) and each 10pmol of upstream/downstream primer, the dNTPs of 2 μ l, 10 * Taq enzyme buffer liquid and 2 μ l 2mM, Taq enzyme (the precious biotech company in Dalian) 1U, moisturizing is to cumulative volume 20 μ l.Behind 94 ℃ of pre-sex change 5min, 94 ℃ of sex change 40Sec, annealing 40Sec, 72 ℃ are extended 40Sec, 25 circulations of increasing, last sample 5 μ l adopt 1% agarose gel electrophoresis to detect.
(SDS-PAGE, Western Blot) and activation analysis (ELISA) are identified in the expression of the structure of embodiment 2.pFUW802s/CEAscFv and anti-CEA single-chain antibody
1.pFUW802s/CEAscFv structure
The gene order of utilizing the reaction of XhoI/EcoRI double digestion to downcut anti-CEA single-chain antibody the soluble-expression carrier (pTCEF/CEAscFv) in the born of the same parents of the anti-CEA single-chain antibody of this laboratory structure is as the external source fragment of following ligation; PFUW802s and pFUW802 are also carried out same endonuclease reaction, reclaim big fragment after, as the carrier segments of following ligation.The gene order of anti-CEA single-chain antibody derives from reference (Koga H.et al., 1990).Endonuclease reaction mixture: the carrier about 1 μ g (10 μ l), 2 μ l, 10 * damping fluid, 1 μ l XhoI and 1 μ l EcoRI, 6 μ l distilled waters.Reaction conditions: 37 ℃ of 2hr.The recovery that enzyme is cut product is with reference to above.
The ligation mixture: 6 μ l pUW802s or pFUW802 enzyme are cut big fragment product, and 20 μ l pTCEF/CEAscFv enzymes are cut the small segment product, 1 μ l T4 dna ligase (the precious biotech company in Dalian), 3 μ l, 10 * T4 dna ligase.16 ℃ connect 2hours.Connect product and be used for transformed into escherichia coli TOP10.Method for transformation is the same.37 ℃ of shaking tables of picking mono-clonal colony inoculation LB-A substratum are cultivated 12hr, adopt the plasmid of Shen, Shanghai energy betting office to extract test kit extraction plasmid in a small amount, because after being connected into anti-CEA single-chain antibody gene sequence, the size of carrier obviously becomes big than pFUW802s or pFUW802, adopts 1% agarose electrophoresis just can identify positive colony.The positive colony carrier is pericentral siphon solinocrine type expression vector pFUW802s/CEAscFv and pFUW802/CEAscFv.
2. the expression of anti-CEA single-chain antibody
Adopt the plasmid of Shen, Shanghai energy betting office to extract test kit extraction plasmid pFUW802s/CEAscFv, pFUW802/CEAscFv, pFUW802s and pFUW802 transformed into escherichia coli HB2151 (available from Pharmacia company) respectively in a small amount, be coated with the LB-A flat board, 37 ℃ of overnight incubation.Picking mono-clonal bacterium colony, inoculation contains 5ml SBAG-2% liquid nutrient medium (35g/ltryptone, 20g/l yeast extract, 5g/l NaCl, and 100 μ g/ml penbritins, pH7.5,2% glucose) in the Boiling tube, 30 ℃ of shaking table overnight incubation, shaking speed 250rpm.1/100 switching contains 5ml SBAG-0.1% liquid nutrient medium/pipe (35g/l tryptone, 20g/l yeast extract, 5g/lNaCl, and 100 μ g/ml penbritins, pH7.5,0.1% glucose) Boiling tube in, 30 ℃ of shaking tables are cultivated 2-3hr, shaking speed 250rpm.The monitoring bacterial growth is to OD
600During=0.8-0.9, add IPTG (isopropyl-) (available from the precious biotech company in Dalian) to final concentration 1mmol/L.Continue 30 ℃ of shaking tables and cultivate 20-24hr, shaking speed 250rpm.
3. salt shock method is extracted colibacillus periplasm chamber protein ingredient
Get 1ml bacterium liquid 1, the centrifugal 20min of 500g collects all thalline, carefully absorbs supernatant, every pipe adds 100 μ l, 1 * TES damping fluid (0.2mol/L Tris-HCl pH8.0,0.5mmol/L EDTA, 0.5mol/L sucrose), add 1/5 * TES that 150 μ l ice precooling behind the mixing, mixing once more, place 30min on ice, carry out the infiltration of salt shock, pericentral siphon chamber protein ingredient is infiltrated.12, the centrifugal 10min of 000g keeps centrifugal supernatant, promptly contains pericentral siphon chamber protein ingredient.Use 500 μ l PBS (8 gram NaCl, 0.2 gram KCl, 1.44 gram Na
2HPO
4With 0.24 gram KH
2PO
4, transfer pH to 7.4 with HCl, be settled to 1 liter) the suspended centrifugal cell precipitation, behind the ultrasonication thalline, 12, centrifugal 10 minutes of 000rpm, centrifugal supernatant is ultrasonic supernatant, and centrifugation is resuspended among the 500 μ l PBS, is ultrasound precipitation.Attention: ultrasonic supernatant contains soluble-expression component in the born of the same parents; Ultrasound precipitation contains inclusion body expresses component.
4. the evaluation of pericentral siphon chamber protein ingredient (SDS-PAGE, Western Blot)
Above-mentioned various component (pericentral siphon chamber protein ingredient, ultrasonic cleer and peaceful ultrasound precipitation) is used to 12%SDS-PAGE and Western-blot identifies.The electrophoretic correlation step of SDS-PAGE is as follows.
(1) preparation of polyacrylamide gel:
(2) processing of sample: an amount of protein sample (general about 30 μ l) and isopyknic 2 * sds gel electrophoresis sample loading buffer (100mmol/L Tris-HCl pH6.8,200mmol/L DTT, 4%SDS, 0.2% tetrabromophenol sulfonphthalein, 20% glycerine) mix, boiling water bath heating 5 minutes, cooling are equipped with goes up sample.
(3) electrophoresis: the preparation electrophoretic buffer (25mmol/L Tris-HCl, pH8.3,0.1%SDS).Get sample on the sample of an amount of volume, constant voltage 90-100V treats that sample enters separation gel, constant voltage 120-150V.
(4) dyeing and decolouring: (0.25g Coomassie brilliant blue R250 is dissolved in (90ml methyl alcohol: water (1:1 is v/v) with the 10ml glacial acetic acid), room temperature dyeing 4-12 hour in 100ml methanol-water-glacial acetic acid solution to adopt Coomassie brilliant blue R250 dyeing.With methanol-water-glacial acetic acid solution decolouring, room temperature is decoloured to having obvious band, gel photograph analytical results (Fig. 3).
If carry out Western blot, sample preparation and SDS-PAGE are the same, use instead and dye the molecular weight of albumen standard in advance.As follows according to concrete operations:
(1) after electrophoresis finishes,, puts into electricity and change damping fluid and soak, cut out the cellulose membrane bigger slightly (Pharmacia company) and in smaller filter paper one coexists electricity commentaries on classics damping fluid, soak than gel with gel clear water rinsing.On electricity commentaries on classics folder, add successively: filter paper-gel-cellulose membrane-filter paper, each interlayer bubble of Ex-all.
(2) electricity changes: after constant current 0.3-0.4A electricity changes 1-2hr, take off tunica fibrosa and gel,
(100mmol/L Tris-Cl, 0.9% (w/v) NaCl pH7.5) clean tunica fibrosa with TBS.
(3) sealing: cellulose membrane is immersed in the confining liquid (2%BSA-TBS), gently jolting 2hr.
(4) adding one resists: wash film three times with TBST (100mmol/L Tris-Cl, 0.9% (w/v) NaCl, pH7.5,0.05-0.1% (V/V) Tween 20), each 5min.Cellulose membrane is immersed in (the 9E10 ascites that contains 1 * 1000 dilution), jolting 1hr gently in the confining liquid.The hybridoma of secretion 9E10 monoclonal antibody is from ATCC (U.S. typical case's culture collection storehouse, American Type Culture collection).Reference experiment handbook (antibody engineering, second edition, Dong Zhiwei, Wang Yan chief editor, Beijing Medical University and combined publication society of China Concord Medical Science University, 2002) preparation 9E10 ascites.
(5) adding two resists: wash membrane method as above.Cellulose membrane is immersed in confining liquid (the HRP mark sheep anti-mouse igg (R﹠amp that contains 1 * 5000 dilution; D company) in, jolting 1hr gently.
(6) colour developing: wash membrane method as above.Add DAB (3,3 '-diamionbenzidene, Sigma product) colour developing liquid, use flushing with clean water when treating colour developing to desirable degree, termination reaction, cellulose membrane keeps in Dark Place after fully washing.Experimental result is seen Fig. 4.
5. the activation analysis of pericentral siphon chamber protein ingredient (ELISA)
Adopt direct ELISA to detect the antigen-binding activity of antibody in pericentral siphon chamber protein ingredient and the ultrasonic supernatant.The ELISA experimental result is seen Fig. 5.The ELISA concrete operations are as follows:
(1) bag quilt: the colon cancer cell SW1116 that collects the abundant CEA of expression is (from U.S. typical case's culture collection storehouse: ATCC, American type culture collection), through after the simple ultrasonication, 12, the centrifugal 5min of 000g collects supernatant, after the process protein concentration is quantitative, concentration with 10 μ g/ml is wrapped quilt, 100 μ l/ holes.Bag is cushioned liquid and (adds Na in every premium on currency
2CO
31.59g, NaHCO
32.93g, transfer pH9.6), 37 ℃ of bags are by 1hr.
(2) after sealing: PBS washes plate 1-2 time, add confining liquid (adding 4% skim-milk or 1%BSA among the PBS) 100 μ l/ holes, 37 ℃ are sealed 1-2hr.
(3) after application of sample: PBS washes plate three times, add doubling dilution (1 *, 2 *, 4 *, 8 *) sample (comprising contrast), hatch 1-2hr for 37 ℃.
(4) add one anti-: after PBST (adding 0.05%Tween-20 in PBS) washed plate three times, with confining liquid 1:1000 dilution 9E10 ascites, 1-2hr was hatched for 37 ℃ in 100 μ l/ holes.
(5) add two anti-: after PBST washes plate three times, with the sheep anti-mouse antibody IgG of confining liquid dilution HRP mark to working concentration.Hatch 1-2hr for 37 ℃.
(6) after colour developing: PBST washes plate 5 times, add colour developing liquid (48.6ml0.1M citric acid, 51.4ml 0.2M Na by 100 μ l/ holes
2HPO
4Add water to 1L, pH5.0 is made into substrate buffer solution.Adding 4mg OPD in the 10ml substrate buffer solution is O-Phenylene Diamine, 15 μ l 30%H
2O
2Be made into colour developing liquid), room temperature lucifuge colour developing 15-30min.
(7) termination reaction: add stop buffer (2M H by 50 μ l/ holes
2SO
4).
(8) read light absorption value at 490nm.
The structure of embodiment 3.pFUW802s/hSLC and the expression of hSLC and evaluation (SDS-PAGE and Western Blot)
Adopt the plasmid of Shen, Shanghai energy betting office to extract test kit extraction plasmid pTCEF/hSLC in a small amount.The gene order of hSLC derives from reference (Nagira M.et al., 1997).Design primer (primer 1:5 ' ATT CTC GAG ATG AAA AAG AAT ATC GCATTT3 ' (SEQ ID NO:6); Primer 2: 5 ' TTC GAA TTC TCG GGC CTT TCGGGG TCT GGC TAC GTT C3 ' (SEQ ID NO:7)) from the gene order of this carrier amplification hSLC, add restriction enzyme site XhoI and EcoRI at the two ends of this sequence simultaneously, PCR reaction mixture: 1 μ l plasmid, each 1 μ l (10pmol) of primer 1 and primer 2,2 μ l pfu, 10 * damping fluid, 2 μ l dNTP (2mmol/L), 0.3 μ l pfu (1U, available from Shen, Shanghai energy betting office), 13 μ l distilled waters.Reaction conditions: 94 ℃ of pre-sex change 3min, 94 ℃ of sex change 30sec, 50 ℃ of annealing 30sec, 72 ℃ are extended 40sec, totally 25 circulations.The PCR product reclaims back (method is the same) through 1% agarose electrophoresis, carries out the reaction of XhoI/EcoRI double digestion simultaneously with carrier pFUW802s.The endonuclease reaction condition is the same.PCR product enzyme is cut the external source fragment of back as ligation, and the carrier enzyme is cut the carrier segments of back as ligation.The ligation condition is the same.
Connect the conversion of product, positive colony is identified, expresses identical with embodiment 2 with the method for identifying.SDS-PAGE and Western blot the results are shown in Figure 6.
Reference:
1.Swartz?JR.Advances?in?Escherichia?coli?production?of?therapeutic?proteins.Curr?OpinBiotechnol.2001?Apr；12(2)：195-201.
2.Ritz?D，Beckwith?J.Roles?of?thiol-redox?pathways?inbacteria.Annu?Rev?Microbiol2001；55：21-48.
3.Missiakas?D.et?al.Protein?folding?in?the?bacterial?periplasm.J.Bacteriol.1997.179：2465-2471.
4.Humphereys?DP，Weir?N，Lawson?A.Co-expression?of?human?disulfide?isomerase(PDI)canincrease?the?yield?of?an?antibody?Fab′fragment?expressed?in?Escherichia?coli.FEBS?Lett.1996，380：194-197；
5.Kurokawa?Y，Yanagi?H，Yura?T.Overexpression?of?protein?disulfide?isomerase?DsbC?stabilizesmultiple-disulfide-bonded?recombinant?protein?produced?and?transported?to?the?periplasm?in?Escherichiacoli.Appl?Environ?Microbiol，2000，66：3960-5；
6.Kurokawa?Y，Yanagi?H，Yura?T.Overproduction?of?bacterial?protein?disulfide?isomerase(DsbC)and?its?modulator(DsbD)markedly?enhances?periplasmic?production?of?human?nerve?growthfactor?in?Escherichia?coli.J?Biol?Chem，2001，276：14393-99；
7.Zhang?Z，Li?ZH，Wang?F.Overexpression?of?DsbC?and?DsbG?Markedly?Improves?Soluble?andFunctional?Expression?of?Single-Chain?Fv?Antibodies?in?Escherichia?coli.Protein?Expr?Purif，2002，6(2)：218-28；
8.Zhang?Z，Song?LP，Fang?M，Wang?F，He?D，Zhao?R，et?al.Production?of?soluble?andfunctional?engineered?antibodies?in?Escherichia?coli?improved?by?FkpA.Biotechniques2003；35(5)：1032-8，41-2.
9.Ramm?K，Pluckthun?A.High?enzymatic?activity?and?chaperone?function?are?mechanisticallyrelated?features?of?the?dimeric?E.coli?peptidyl-prolyl-isomerase?FkpA.J?Mol?Biol，2001，310：485-498；
10.Bothman?H，Pluckthun?A.The?periplasmic?Escherichia?coli?peptidylprolyl?cis，trans-isomerase?FkpA.I.Increased?functional?expression?of?antibody?fragments?with?and?without?cis-prolines.J?Biol?Chem.2000，275：17100-17105；
11.Ramm?K，Pluckthun?A.The?periplasmic?Escherichia?coli?peptidylprolyl?cis，trans-isomeraseFkpA.II.Isomerase-independent?chaperone?activity?in?vitro.J?Biol?Chem.2000，275：17106-17113；
12.Arie?JP，Sassoon?N，Betton?JM.Chaperone?function?of?FkpA，a?heat?shock?prolylisomerase，in?the?periplasm?of?Escherichia?coli.Mol?Microbiol.2001，39：199-210；
13.Mergulhao?FJ，Monteiro?GA，Larsson?G，Sanden?AM，Farewell?A，Nystrom?T，Cabral?JM，Taipa?MA.Medium?and?copy?number?effects?on?the?secretion?of?human?proinsulin?in?Escherichia?coliusing?the?universal?stress?promoters?uspA?and?uspB.Appl?Microbiol?Biotechnol.2003?Jun；61(5-6)：495-501.
14.Mergulhao?FJ，Monteiro?GA，Larsson?G，Bostrom?M，Farewell?A，Nystrom?T，Cabral?JM，Taipa?MA.Evaluation?of?inducible?promoters?on?the?secretion?of?a?ZZ-proinsulin?fusion?protein?inEscherichia?coli.Biotechnol?Appl?Biochem.2003?Aug；38(Pt1)：87-93.
15.Winter?J，Neubauer?P，Glockshuber?R，Rudolph?R.Increased?production?of?human?proinsulinin?the?periplasmic?space?of?Escherichia?coliby?fusion?to?DsbA.J?Biotechnol.2001?Nov?30；84(2)：175-85.
16.Abrahmsen?L，Moks?T，Nilsson?B，Uhlen?M.Secretion?of?heterologous?gene?products?to?theculture?medium?of?Escherichia?coli.Nucleic?Acids?Res.1986?Sep?25；14(18)：7487-500.
17.Uhlen?M，Abrahmsen?L.Secretion?of?recombinant?proteins?into?the?culture?medium?byEscherichia?coli?and?Staphylococcus?aureus.Biochem?Soc?Trans.1989?Apr；17(2)：340-1.
18.Smith?DB，Johnson?KS.Single-step?purification?of?polypeptides?expressed?in?Escherichia?colias?fusions?with?glutathione?S-transferase.Gene.1988?Jul?15；67(1)：31-40.
19.Sharrocks?AD.A?T7?expression?vector?for?producing?N-and?C-terminal?fusion?proteins?withglutathione?S-transferase.Gene.1994?Jan?28；138(1-2)：105-8.
20.LaVallie?ER，DiBlasio-Smith?EA，Collins-Racie?LA，Lu?Z，McCoy?JM.Thioredoxin?andrelated?proteins?as?multifunctional?fusion?tags?for?soluble?expression?in?E.coli.Methods?Mol?Biol.2003；205：119-40.
21.Koga?H，Kanda?H，Nakashima?M，WatanabeY，Endo?K，Watanabe?T.Mouse-human?chimericmonoclonal?antibody?to?carcinoembryonic?antigen(CEA)：in?vitro?and?in?vivo?activities.Hybridoma1990；9(1)：43-56.
22.Nagira?M，Imai?T，Hieshima?K，Kusuda?J，Ridanpaa?M，Takagi?S，Nishimura?M，Kakizaki?M，Nomiyama?H，Yoshie?O.Molecular?cloning?of?a?novel?human?CC?chemokine?secondary?lymphoid-tissuechemokine?that?is?a?potent?chemoattractant?for?lymphocytes?and?mapped?to?chromosome?9p13.J?BiolChem.1997?Aug?1；272(31)：19518-24.
23. open defend the country, Liu Xifu, Wang Yong etc., the structure in phage oblatio single-chain antibody expression vector and mouse nonspecific antibody storehouse. Acta Genetica Sinica .1999.26 (2): 99-106.
SEQUENCE?LISTING
＜110〉Beijing Anbote Gene Engineering Co., Ltd.
＜120〉a kind of colibacillus periplasm solinocrine type expression vector
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Claims (5)
1. pericentral siphon solinocrine type expression vector of expressing foreign protein, it is characterized in that it is a kind of co-expression carrier, the gene and the gene that is connected with the foreign protein of signal peptide that contain short folding associated protein, the gene of wherein said short folding associated protein and the gene of described foreign protein are under the control of same transcripting promoter, or under the control of different transcripting promoters, described short folding associated protein is a FK506 binding protein peptide acyl prolyl cis-trans isomerase, and described signal peptide is the polygalacturonase signal peptide of sequence shown in SEQ IDNO:1.
2. the expression vector of claim 1, it is expression vector pFUW802s as shown in Figure 3.
3. the expression vector of claim 1, it is to utilize the XhoI/EcoRI restriction enzyme site that anticancer embryonal antigen single-chain antibody gene is cloned into pFUW802s as shown in Figure 3 and the pFUW802s/CEAscFv that obtains.
4. host cell that contains any one described carrier among the claim 1-3, it is intestinal bacteria.
5. express the method for foreign protein at the pericentral siphon solinocrine for one kind, it is included in and utilizes each described expression vector of claim 1-3 to express the step of the gene of described foreign protein in the described host cell of claim 4.
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Non-Patent Citations (5)
| Title |
|---|
| WO、A2，03018771 2003.03.06 |
| 单链抗体在大肠杆菌中的分泌型表达. 刘德新等.军医进修学院学报，第23卷第3期. 2002 |
| 单链抗体在大肠杆菌中的分泌型表达. 刘德新等.军医进修学院学报，第23卷第3期. 2002 * |
| 相容性溶质支持下重组免疫毒素的周质腔可溶表达及葡萄糖对其表达的影响. 任学明等.遗传，第25卷第1期. 2003 |
| 相容性溶质支持下重组免疫毒素的周质腔可溶表达及葡萄糖对其表达的影响. 任学明等.遗传，第25卷第1期. 2003 * |
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