CN103173478B - A kind of preparation method carrying the anti-human CD3 single-chain antibody of PTD - Google Patents
A kind of preparation method carrying the anti-human CD3 single-chain antibody of PTD Download PDFInfo
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Abstract
The invention discloses a kind of carry PTD anti-human CD3 single-chain antibody (being called for short PTD-CD3) renaturation and the preparation method of purifying.First will form PTD-CD3 single-chain antibody solubilization of inclusion bodies in urea solution, and adopt silica matrix efficient hydrophobic interaction chromatograph (HPHIC) post or chromatographic cake to carry out PTD-CD3 single-chain antibody renaturation and Simultaneous purification.Obtaining mass recovery to the method optimize chromatography condition and HPSEC desalination is 46%, and purity is the PTD-CD3 single-chain antibody of 96%, and competition binding restraining effect confirms that PTD-CD3 has the biological activity of natural CD3 single-chain antibody.
Description
Technical field
The present invention relates to a kind of preparation method carrying the anti-human CD3 single-chain antibody of PTD, belong to biological technical field.
Background technology
CD3 is the lymphocytic a kind of differentiation antigen of T, by γ, δ, ε, P
28article 4, the polymer of polypeptide chain composition, it forms TCR-CD3 mixture together with T cell antigen acceptor (TCR), plays an important role in T cell identification antigen and reactivation process.At the beginning of 80, Ortho company of the U.S. uses hybridoma technology, develop the monoclonal antibody (anti-CD3 Monclonalantibody, anti-CD3McAb) of human lymphocyte surface antigen, because its many unique immune regulates the active great interest causing people.To the preparation of the transformation of murine antibody, full-length human antibody, the mutant antibodies of screening high-affinity and and other biologically active substance be cross-linked to form difunctional structure etc. and all become a reality, even do not need can obtain antibody molecule gene by animal immune, the research of genetic engineering antibody is goed deep into being widely used of field such as diagnosis, treatment, bio-guides, and achieves some stem-winding results.
Nexin transduction domain (PTD) is one section of polypeptide fragment of the trans-activator TAT coded by human immunodeficiency virus (HIV)-1, has unique transmembrane transport mode, and be characterized in that transduction rate is fast, efficiency is high.The multiple biomacromolecules such as polypeptide, oligonucleotide, antibody, enzyme can be imported the target cell of 100% by PTD in a short time.If the gene of peptide section-PTD of specific function and the variable region gene selectivity of anti-cd 3 antibodies gene heavy chain and light chain are merged, can obtain carrying PTD AntiCD3 McAb single-chain antibody gene, make CD3 enter faster penetration cell, play its biological function.
Summary of the invention
The object of this invention is to provide a kind of preparation method adopting efficient hydrophobic interaction chromatography one step to realize carrying anti-human CD3 single-chain antibody (PTD-CD3) renaturation of PTD and purifying.
Implementation procedure of the present invention is as follows:
Carry a preparation method for the anti-human CD3 single-chain antibody of PTD, comprise the following steps:
(1) natural anti-cd 3 antibodies gene and peptide section-PTD gene fusion obtain carrying PTD AntiCD3 McAb single-chain antibody gene, will carry PTD AntiCD3 McAb single-chain antibody gene and insert
e.coliexpression Vectors pBV220 is expressed, and obtains anti-human CD3 single-chain antibody (PTD-CD3) recombinant bacterium carrying PTD;
(2) PTD-CD3 single-chain antibody recombinant bacterium exists
e.coliin expression product be inclusion body containing PTD-CD3, by solubilization of inclusion bodies in 6.0 ~ 8.0 mol/L urea solutions, use structural formula (I) or the HPHIC medium shown in (II) to carry out renaturation and purifying, collect chromatographic fraction, obtain the PTD-CD3 of renaturation and purifying;
（I）
Wherein n=10 ~ 25
（II）
bare substrate is aperture is 20-30 nm; Particle diameter is 5-10
μthe macroporous silica gel microballoon of m;
Chromatographic condition: mobile phase A liquid is 2 ~ 3 mol/L (NH
4)
2sO
4, 20 ~ 50 mmol/L KH
2pO
4, pH 7 ~ 8; Mobile phase B liquid is 20 ~ 50 mmol/L KH
2pO
4, pH 7 ~ 8; Flow velocity is 1.0 ~ 5.0 mL/min; Adopt 0-100% B linear gradient wash-out; Determined wavelength 280 nm;
(3) be further purified by PTD-CD3 single-chain antibody Size Exclusion Chromatograph SEC, Size Exclusion Chromatograph SEC medium is Superdex75R.
In above-mentioned steps (1), peptide section-PTD sequence is GRKKRRQRRRPPQ, and the PTD-CD3 single-chain antibody recombinant bacterium of acquisition is pBV220/PTD-CD3/DH5 α.
In above-mentioned steps (2), the inclusion body containing PTD-CD3 dissolves with strong denaturant 6.0 ~ 8.0 mol/L urea, places 12 more than h and is separated, and obtains containing PTD-CD3 inclusion body sex change extract.
In above-mentioned steps (2), in the efficient hydrophobic interaction chromatograph stationary phase shown in structural formula (I), n equals 20.
In above-mentioned steps (2), efficient hydrophobic interaction chromatograph post is respectively 10 × 20 mm I.D. pie analysis modes or 10 × 50 mm I.D. half preparative chromatography posts, and the pressure loading chromatographic column is voluntarily greater than 40 MPa.Described 10 × 20 mm I.D. pie analysis modes or 10 × 50 mm I.D. half preparative chromatography post referenced patent methods preparation (ZL9210272.3, US 7,208,085 B2, EP1 396 721 B1).
Advantage of the present invention: (1) obtains one after utilizing the gene fusion of the peptide section-PTD of natural anti-cd 3 antibodies gene and specific function and carries PTD AntiCD3 McAb single-chain antibody gene, this fusion gene obtains high expression in intestinal bacteria, carry out the confirmation of competition binding Inhibition test by after expression product renaturation in a small amount and purifying, the anti-human CD3 single-chain antibody of this PTD has CD3 affinity.(2) analysis mode and semipreparative efficient hydrophobic interaction chromatograph cake one step achieve renaturation and the purifying of PTD-CD3 single-chain antibody, chromatographic stationary phases aglucon, moving phase composition, pH value, buffers combinations, gradient, sample size and flow velocity are optimized, mass recovery reaches 46%, and purity is 89%; (2) be further purified with Size Exclusion Chromatograph SEC and obtain the PTD-CD3 that purity is 96%; (3) the method can prepare PTD-CD3 by fast purifying, is easy to reach large-scale production.
Accompanying drawing explanation
Fig. 1 is with stationary phase aglucon be PEG600 HPHIC renaturation and purifying color atlas
In figure, X-coordinate is retention time (time), and ordinate zou is wavelength 280nm place ultraviolet absorption value (mAU);
Fig. 2 is with stationary phase aglucon be PEG800 HPHIC renaturation and purifying color atlas
In figure, X-coordinate is retention time (time), and ordinate zou is wavelength 280nm place ultraviolet absorption value (mAU);
Fig. 3 is with stationary phase aglucon be phenyl HPHIC renaturation and purifying color atlas
In figure, X-coordinate is retention time (time), and ordinate zou is wavelength 280nm place ultraviolet absorption value (mAU);
After Fig. 4 HPHIC renaturation and Simultaneous purification, the SDS-PAGE of PTD-CD3 single-chain antibody analyzes
1, the product of Size Exclusion Chromatograph SEC purifying; 2, the product of HPHIC renaturation and purifying;
The color atlas of Fig. 5 Size Exclusion Chromatograph SEC purifying
In figure, X-coordinate is retention time (time), and left ordinate zou is wavelength 280nm place ultraviolet absorption value (mAU), and right ordinate zou is conductivity variations (ms/cm);
Fig. 6 HPRPLC is to purifying PTD-CD3 purity check
In figure, X-coordinate is retention time (time), and ordinate zou is wavelength 280nm place ultraviolet absorption value (mAU);
The competition binding the Inhibitory Effects of Fig. 7 PTD-CD3 single-chain antibody
In figure, X-coordinate is PTD-CD3 concentration (mg/mL), and ordinate zou is competition binding inhibiting rate (%).
Embodiment
The invention will be further described by the following examples, and following Examples is used for illustrating but not limiting the present invention.
Embodiment 1
Be 30 nm with aperture; Particle diameter is 10
μhigh-purity macroporous silica gel of m is as bare substrate.First carry out acid treatment, by the hydrochloric acid 120 DEG C of silica gel 1:1 backflow 3 ~ 4 h, cooling, then uses distilled water repetitive scrubbing, until solution is aobvious neutral, and dried in vacuo overnight.Silica gel γ good for acid treatment-glycidoxypropyl trimethoxy silane (TM-560) is activated, under 90 DEG C of conditions, slowly drip TM-560 appropriate, react 2 h with silica gel, then use water, methyl alcohol, water washing clean successively, vacuum-drying, obtains epoxy group(ing) silica gel.Then in the three-necked bottle of 1000 mL, add 100g epoxy group(ing) silica gel, add 500 mL dioxane, the boron trifluoride diethyl etherate of 200 mL48% and 200 mL phenylcarbinols or 250 mL polyethylene glycol-800 simultaneously, ultrasonic 5min, stirring at normal temperature 2h, the HPHIC filler of phenyl type and PEG800 type is dried and obtained to product acetone, methyl alcohol, water washing totally.Characterized by ultimate analysis, infrared, the HPHIC filler of XPS to phenyl type and PEG800 type, confirm its structure as described in structural formula (I) or (II).
Embodiment 2
1, recombinant bacterium is cultivated and fermentation
Transformed by intestinal bacteria (E.coli) the DH5 α expression plasmid of anti-human CD3 single-chain antibody (the being called for short PTD-CD3) gene carrying PTD containing coding and expression, this plasmid is loaded with the P transcribing PTD-CD3
lp
rpromotor, is loaded with anti-ampicillin gene, and expressed PTD-CD3 product exists with insoluble inclusion bodies.PTD-CD3 transforms bacterial classification and is containing yeast extract 5g/L, Tryptones 10g/L, NaCl 10g/L, and in the seed culture medium of penbritin l00mg/L, shaking table 30 DEG C of 150r/min activation culture 12h, the 5L fermentor tank of 4.0L substratum is equipped with in culture inoculation.Substratum consists of yeast extract 5g/L, Tryptones 10g/L, phosphate buffered saline buffer 50 mmol/L, magnesium sulfate 4 mmol/L, with front adding 50% glycerine.In tank, dissolved oxygen is by dissolved oxygen electrode on-line checkingi, regulates mixing speed and ventilation flow rate, controls dissolved oxygen and is greater than 30% saturated oxygen concentration all the time.PH also by pH electricity level on-line checkingi, fills into 5.0 mol/L ammoniacal liquor control ph by peristaltic pump and is not less than 6.5.Be warming up to 42 ± 1 DEG C of induction 5h after 30 ± 1.5 DEG C of cultivation 6-8h after inoculation, results culture, uses 8000r/min centrifugation, receives about 40g bacterial sediment thing.
2, the recovery of inclusion body and dissolving
The thalline of fermentation is suspended in damping fluid I(0.2 mol/L PBS according to 1:5 (g/mL, m/v), 1 m mol/L EDTA, pH7.4) in, 4 DEG C, 8000 g × 10 min are centrifugal abandons supernatant liquor.Add damping fluid I according to volume ratio 1:5 equally, be placed in ice bath ultrasonication 30 min, 4 DEG C, 17,000 g × 15 min is centrifugal, abandon supernatant liquor.Again by throw out in 1:5 ratio with damping fluid I suspend, 4 DEG C, 17, the centrifugal bacterial sediment thing of 000 g × 15 min, can repeat 2-3 time, removing foreigh protein removing.Purity through the PTD-CD3 inclusion body of thick purifying is greater than 75%.The inclusion body of above-mentioned preparation adds 8 mol/L ureas or 7 mol/L, Tris-HCl 10 mmol/L in the ratio of 1:15, the solution of pH8.0, under the condition of 4 DEG C, can agitation as appropriate spend the night, next day, 4 DEG C, 17,000 g × 15 min collected by centrifugation supernatant liquor, for subsequent use as sex change extract.
3, the chromatographic cake renaturation of different aglucon Simultaneous purification PTD-CD3
8 mol/L Urea denaturation extract 1.0 mL direct injection are prepared spectrum cake to half of filling hydrophobic chromatographic stuffing, the aglucon of filler is respectively PEG600, PEG800, phenyl, 10 mm × 20mmI.D.), in flow velocity 2.0 ml/min, wavelength 280 nm condition down to using 100%A(3.0 mol/L (NH
4)
2sO
4, 50m mol/L KH
2pO
4, pH7.0) and balance, then non-linear gradient elution 20 min is to Mobile phase B (50m mol/LKH
2pO
4, pH7.0) and be 100%, then continue wash-out 5 min with B liquid, chromatographic column is regenerated, finally by 100% mobile phase A, chromatographic column is balanced again, to form a complete chromatographic process.After three kinds of estimation for ligand screening with specific phages, be that the mass recovery of the PTD-CD3 that the stationary phase renaturation of PEG800 and purifying obtain is the highest with aglucon, can reach 46%, purity is 89%.Color atlas is shown in Fig. 1-3 respectively, and electrophoresis result is shown in second in Fig. 4.
4, size exclusion chromatography purifying PTD-CD3
By the PTD-CD3 Superdex75 after HPHIC renaturation and purifying
r(Pharmacia Products), flow velocity 2 mL/min, level pad I (containing Tris-HCl10 mmol/L, pH7.0) pre-equilibration used in advance by this post.UV-detector wavelength 280 nm detects elutriant, and through each step purifying, final product purity has brought up to 96%.First in Fig. 5 is shown in electrophoretic analysis.
5, the purity check of renaturation and purifying PTD-CD3
HP-RPLC moving phase: A liquid: pure water+0.1% TFA; B liquid: methyl alcohol+0.1% TFA.The PTD-CD3 solution sample introduction getting final purifying to by the chromatographic column (100 × 4.0 mm, i.d.) that 50 %A liquid balance, with 0-100 %B linear gradient elution 10 min, extend 5 min, flow velocity 1.0mL/min, determined wavelength 280 nm, color atlas is shown in Fig. 6.
6, the competition binding restraining effect of renaturation and purifying PTD-CD3
Be separated human peripheral lymphocyte with the lymphocyte separation medium bought, add 96 orifice plates, cell concn is 1 × 10
5/ hole, add the PTD-CD3 fusion rotein of doubling dilution and the AntiCD3 McAb single-chain antibody of purifying, wherein by anti-human CD3 monoclonal antibody as positive control, anti-human alpha-fetoprotein (AFP) monoclonal antibody contrasts as irrelevant antibody, after 4 DEG C of insulation 30 min, adds FITC mark mouse-anti people CD3 monoclonal antibody and is incubated 30 min, ice-cold PBS washing, fix according to 10mL/L (v/v) paraformaldehyde, use cells were tested by flow cytometry average fluorescent strength, calculate inhibiting rate.7 be the results are shown in Figure to the competition binding restraining effect of PTD-CD3 single-chain antibody.
Claims (1)
1. carry a preparation method for the anti-human CD3 single-chain antibody of PTD, comprise the following steps:
(1) natural anti-cd 3 antibodies gene and peptide section-PTD gene fusion obtain carrying PTD AntiCD3 McAb single-chain antibody gene, will carry PTD AntiCD3 McAb single-chain antibody gene and insert
e.coliexpression Vectors pBV220 is expressed, and obtains the anti-human CD3 single-chain antibody recombinant bacterium carrying PTD,
Above-mentioned peptide section-PTD sequence is GRKKRRQRRRPPQ, and the PTD-CD3 single-chain antibody recombinant bacterium of acquisition is pBV220/PTD-CD3/DH5 α;
(2) PTD-CD3 single-chain antibody recombinant bacterium exists
e.coliin expression product be inclusion body containing PTD-CD3, by solubilization of inclusion bodies in 6.0 ~ 8.0 mol/L urea solutions, structural formula (I) or the HPHIC medium shown in (II) is used to carry out renaturation and purifying, collect chromatographic fraction, obtain the PTD-CD3 of renaturation and purifying, the inclusion body containing PTD-CD3 dissolves with strong denaturant 6.0 ~ 8.0 mol/L urea, places separation in more than 12 hours, obtain containing PTD-CD3 inclusion body sex change extract
（I）
Wherein n=20
（II）
bare substrate is aperture is 20-30 nm, and particle diameter is 5-10
μthe macroporous silica gel microballoon of m;
Chromatographic condition: mobile phase A liquid is 2 ~ 3 mol/L (NH
4)
2sO
4, 20 ~ 50 mmol/L KH
2pO
4, pH 7 ~ 8; Mobile phase B liquid is 20 ~ 50 mmol/L KH
2pO
4, pH 7 ~ 8; Flow velocity is 1.0 ~ 5.0 mL/min; Adopt 0-100% B linear gradient wash-out, determined wavelength 280 nm,
Efficient hydrophobic interaction chromatograph post is respectively 10 × 20 mm I.D. pie analysis modes or 10 × 50 mm I.D. half preparative chromatography posts, and the pressure of filling chromatographic column is greater than 40 MPa;
(3) be further purified by PTD-CD3 single-chain antibody Size Exclusion Chromatograph SEC desalination, Size Exclusion Chromatograph SEC medium is Superdex75R.
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| CN101220081A (en) * | 2008-01-28 | 2008-07-16 | 江苏吴中医药集团有限公司苏州中凯生物制药厂 | Extraction and purification process for recombinant protein |
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| CN101220081A (en) * | 2008-01-28 | 2008-07-16 | 江苏吴中医药集团有限公司苏州中凯生物制药厂 | Extraction and purification process for recombinant protein |
Non-Patent Citations (2)
| Title |
|---|
| "高效疏水相互作用色谱法复性与同时纯化重组人Flt3配体的包涵体蛋白质";贾佳 等;《Chinese Journal of Chromatography》;20100630;第28卷(第6期);第536页左栏第2段，第536页右栏最后1段，第537页左栏最后1段-右栏第1段，537页右栏最后1段 * |
| 王骊丽 等.C26 T细胞毒性的抗人CD3单链抗体的制备.《全国生物医药色谱交流会(2008)论文集》.2008,第139-140页. * |
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