CN102464717A - Preparation method of human antinuclear antibody - Google Patents

Abstract

The invention discloses a preparation method of a human antinuclear antibody, comprising the following steps: collecting single nuclear cells of peripheral blood of a patient with the autoimmune disease, extracting the total RNA (Ribonucleic Acid), and carrying out reverse transcription to obtain a cDNA (Complementary Deoxyribonucleic Acid) library; amplifying a light chain k,1 gene and an immunoglobulin molecule heavy chain Fd gene by taking the acquired cDNA library as a template through a PCR (Polymerase Chain Reaction) method; cloning the light chain k,1 gene into a pComb3Hss carrier to construct a light chain library; loading the heavy chain Fd gene into a carrier to construct and finish a combinatorial library; transforming the obtained recombinant plasmid into escherichia coli XL1-Blue, infecting by using a helper phage M12KO7, and expressing the random combinatorial library on the surface of a filamentous phage to finish phage surface display; and purifying the Fab segment of the human antinuclear antibody by utilizing affinity chromatography. By using the preparation method, the defects that the random combinatorial antibody library has strong randomness, the screening workload is large and a specific antibody is not easy to obtain are overcome, and the specific antibody can be directly screened from a phage antibody library; and in addition, the method is simple and feasible, and the experimental period is shortened.

Description

A kind of preparation method of humanized's antinuclear antibody

Technical field

The invention belongs to biochemical field, particularly a kind of preparation method of humanized's antinuclear antibody.

Background technology

Tumour is the biggest threat of current mankind health and life, and incidence is growing on and on, and mortality ratio has leapt to first of all sick kinds, mainly adopts means such as operation, radiotherapy, chemotherapy to treat, but still can't obtain the curative effect of satisfaction.Tumor necrosis treatment is a kind of new therapy that rises recently; The category that belongs to RIT; Its ultimate principle is to utilize the discontinuity of the CMEC of tumor tissues, the high-permeability characteristics of the imperfection of basilar membrane and tumor cell membrane, and application can be carried effluent matter extremely with intracellular nucleic antigen composition bonded targeted molecular; Be detained or be positioned in the tumor tissues, cause the specific killing of tumour.The targeted molecular that disengages after oncocyte kills and wounds can get into adjacent tumor tissue again and cause further and kill and wound, and makes so extremely that the knurl scope constantly enlarges, and is dead until healthy tissues.The key of this therapy be obtain can with intracellular nucleic antigen composition specificity bonded antibody; Method commonly used at present is to obtain the monoclonal antibody to the neoplasm necrosis tissue through people's murine hybridoma technology, for example the chimeric monoclonal antibody of neoplastic cell nuclei people mouse (chTNT) of Peregrine exploitation.But, use this legal system and be equipped with monoclonal antibody and be used for RIT and have following defective: the first, still there is comparatively serious host's antibody response in it as the chimeric monoclonal antibody of a kind of mouse/people, should not repeatedly use; The second, technology of preparing is more numerous and diverse, and the big requirement of difficulty is high, and success ratio is low; The 3rd, the monoclonal antibody molecular weight that obtains at present is big, and tissue penetration property is relatively poor, is unfavorable for bringing inside tumor into killing effluent matter; The 4th, be neoplasm necrosis tissue because it is directed against, antigen component is complicacy comparatively, so its specificity is relatively poor.

Summary of the invention

The objective of the invention is to overcome the defective that exists in the prior art; The preparation method of humanized's monoclonal anti antinuclear antibodies that a kind of method is easy relatively, input cost is less, success ratio is high is provided, and prepared humanized's monoclonal anti antinuclear antibodies can better be applied to tumor necrosis treatment.

A kind of preparation method of humanized's antinuclear antibody is characterized in that may further comprise the steps:

(a) collect the autoimmune disease peripheral blood mononuclear cells, extracted total RNA, the cDNA library is obtained in reverse transcription;

(b) be masterplate with the cDNA library that obtains, PCR method amplification light chain κ, λ gene and immunoglobulin molecules heavy chain Fd gene;

(c) the pComb3Hss plasmid is gone in light chain κ, λ gene clone, make up the light chain library;

(d) heavy chain Fd gene is written into step c gained plasmid, makes up and accomplish combinatorial library;

(e) with steps d gained recombinant plasmid transformed intestinal bacteria XL1-Blue, infect with helper phage M13KO7, combinatorial library at random is expressed in the filobactivirus surface, accomplishes phage display;

(f) take turns naughty sieve through 4; Humanized's antinuclear antibody Fab phage antibody library that enrichment has made up; Indirect elisa method identifies that 4 take turns naughty sieve back antinuclear antibody Fab phage antibody, extracts the phagemid dna of positive colony, excision gIII gene fragment; From connecting back transformed into escherichia coli XL1-Blue, with IPTG abduction delivering solubility humanized antinuclear antibody Fab fragment; Using indirect elisa method and fluorescent immune method identifies expression product;

(g) utilize affinity chromatography column purification humanized antinuclear antibody Fab fragment.

Further, in the autoimmunization patient blood titre of antinuclear antibody greater than 1: 10000 (use IIF detect).

Further, among the step b, the primer sequence that the amplification of PCR method is used is shown in SEQ ID No.1～SEQ ID No.8:

1. variable region of heavy chain 5 ' end primer VH4f:

5′-CAGGTGCAGCTGCTCGAGTCGGG-3′；(SEQ?ID?No.1)

2. variable region of heavy chain 5 ' end primer VH1a:

5′-CAGGTGCAGCTCGAGCAGTCTGGG-3′；(SEQ?ID?No.2)

3. heavy chain IgG1 (Fd) 3 ' holds primer CG1Z:

5′-GCATGTACTAGTTTTGTCCCAAGATTTGGG-3′；(SEQ?ID?No.3)

4. heavy chain IgG3 (Fd) 3 ' holds primer CG3Z:

5′-TGTGTGACTAGTGTCACCAAGTGGGGTTTT-3′；(SEQ?ID?No.4)

5. light chain (κ) variable region 5 ' end primer V κ 3a:

5′-GAAATTGAGCTCACGCAGTCTCCA-3′；(SEQ?ID?No.5)

6. light chain (λ) variable region 5 ' end primer VL4:

5′-TCTGAAGAGCTCCAGGACCCTGTTGTGTCTGTG-3′；(SEQ?ID?No.6)

7. light chain (κ) constant region 3 ' end primer C κ 1Z:

5′-GCGCCGTCTAGAAACACTCTCCTCTGTTGAAG

CTCTTTGTGACGGATCTCAG-3′；(SEQ?ID?No.7)

8. light chain (λ) constant region 3 ' end primer CL2:

5′-CGCCGTCTAGAACTATGAACATTCTGTAGG-3′；(SEQ?ID?No.8)。

The present invention adopts phage antibody library technique; Collect autoimmune disorder peripheral blood of patients mononuclearcell, extract RNA, obtain the cDNA library; And make up humanized's antinuclear antibody Fab fragment combination library in view of the above, rely on phage display technique to obtain corresponding phage antibody library.Obtain positive colony through the screening of immobilization antigen absorption sieve method, and further realize the segmental expression of solubility antinuclear antibody Fab.The present invention selects autoimmune disorder peripheral blood of patients mononuclearcell for use, helps obtaining to be rich in the cDNA library of antinuclear antibody gene, has improved the success ratio of screening acquisition high-affinity positive colony.The antinuclear antibody Fab fragment that the present invention makes up has heavy chain Fd gene and complete light chain gene, and this small molecular antibody has the activity of antibody, and size is 1/3 of complete IgG, and tissue penetration is stronger relatively; In addition, because of it does not contain the Fc section, molecular weight is little, so immunogenicity is low, more is applicable to tumor necrosis treatment.The present invention adopts the double-stranded DNA antigen antagonist storehouse of solid-phase coating to screen, and it is active that the antibody of acquisition has the specific anti double-stranded DNA, helps to reduce the toxic side effect of tumor necrosis treatment.The present invention uses phage antibody library technique; Phage surface submission technology is introduced the structure of antibody library; Overcome the shortcoming that makes up at random that antibody library randomness is strong, the screening operation amount big and be not easy to obtain specific antibody; Can from phage antibody library, directly filter out specific antibody, method is simple, has shortened experimental period greatly.

Description of drawings

Fig. 1 is the inventive method schema.

Fig. 2 is the pComb3Hss structural representation.

Embodiment

Shown in Figure 1 for preparing method's of the present invention schema.

(1) makes up phage antibody library

1, separates PMNC

(1) gets antinuclear antibody positive patient peripheral blood 3ml, place the anticoagulant heparin pipe;

(2) saline water dilution in 1: 1 whole blood;

(3) in the 10ml of cleaning centrifuge tube, add the 3ml lymphocyte separation medium, the whole blood after the dilution carefully is added on the parting liquid liquid level;

(4) centrifugal, 2000rpm, 20 minutes;

(5) draw buffy coat;

(6) washing of PBS liquid is 2 times;

(7) adding PBS liquid is resuspended, and-80 ℃ of refrigerators in microscopically cell numeration back are preserved.

2, extract total RNA

(1) from PMNC, extracts total RNA with the centrifugal cell total rna extraction agent of a small amount of post.The centrifugal supernatant of abandoning of the PMNC that separating, washing is frozen adds 1ml RL liquid, lashes 5 times with suspended sample with pipettor immediately, static 5 minutes of room temperature.

(2) will digest good cell pyrolysis liquid and be drawn onto during 1.5ml EP that a DEPC handled manages, and add the chloroform 0.2ml that newly opens, and firmly put upside down centrifuge tube, room temperature is static behind the mixing makes it layering.Centrifugal 5 minutes of 12000g speed carefully pipettes in the 1.5ml centrifuge tube that the water item handled to another DEPC.

(3) add isopyknic Virahol of newly opening, thoroughly behind the mixing, take out 750 μ l and move into adsorption column, centrifugal 30 seconds, outwell the liquid in the collection tube, adsorption column is put back in the collection tube.Remaining liq is all moved in the adsorption column, operate equally.

(4) in adsorption column, add 500 μ l RP liquid, centrifugal 30 seconds.Adsorption column is moved in the clean collection tube.

(5) add 500 μ l W3 liquid in the adsorption column, after static 1 minute centrifugal 15 seconds, same method was washed once more.Centrifugal 1 minute.

(6) adsorption column is put into 1.5ml centrifuge tube (DEPC handled).Add 30 μ l pure water in adsorption film central authorities, room temperature after static 1 minute centrifugal 1 minute.With carrying out next step experiment or be stored in-70 ℃ after the RNA evaluation that obtains.

3, cDNA first chain is synthetic

Get 1 μ g RNA and do the rt template, Oligo (d) T 1 μ l does reverse transcriptase primer, adds DEPC water to TV 12 μ l, mixing; 70 ℃ of sex change 5min, ice bath cooling rapidly adds 5 * damping fluid, 4 μ l, RNase suppressor factor 1 μ l successively; DNTP (10mMeach) 2 μ l, mixing is hatched 5min for 37 ℃, adds AMV reversed transcriptive enzyme 1 μ l; 37 ℃ of reverse transcription 60min, 70 ℃ of 10min termination reactions, cooled on ice.

4, be template with rt cDNA, according to primer SEQ ID No.1～SEQ ID No.8, with RT-PCR amplification heavy chain and κ, lambda light chain gene.

1. variable region of heavy chain 5 ' end primer VH4f:

5′-CAGGTGCAGCTGCTCGAGTCGGG-3′；(SEQ?ID?No.1)

2. variable region of heavy chain 5 ' end primer VH1a:

5′-CAGGTGCAGCTCGAGCAGTCTGGG-3′；(SEQ?ID?No.2)

3. heavy chain IgG1 (Fd) 3 ' holds primer CG1Z:

5′-GCATGTACTAGTTTTGTCCCAAGATTTGGG-3′；(SEQ?ID?No.3)

4. heavy chain IgG3 (Fd) 3 ' holds primer CG3Z:

5′-TGTGTGACTAGTGTCACCAAGTGGGGTTTT-3′；(SEQ?ID?No.4)

5. light chain (κ) variable region 5 ' end primer V κ 3a:

5′-GAAATTGAGCTCACGCAGTCTCCA-3′；(SEQ?ID?No.5)

6. light chain (λ) variable region 5 ' end primer VL4:

5′-TCTGAAGAGCTCCAGGACCCTGTTGTGTCTGTG-3′；(SEQ?ID?No.6)

7. light chain (κ) constant region 3 ' end primer C κ 1Z:

5′-GCGCCGTCTAGAAACACTCTCCTCTGTTGAAGCTCTTTGTGACGGATCTCAG-3′；(SEQ?ID?No.7)

8. light chain (λ) constant region 3 ' end primer CL2:

5′-CGCCGTCTAGAACTATGAACATTCTGTAGG-3′；(SEQ?ID?No.8)。

PCR reaction system 25 μ l, cDNA 0.5 μ l wherein, upstream primer 0.25 μ l downstream primer 0.25 μ l, Taq enzyme 0.2 μ l dNTP2 μ l 10 * buffer 2.5 μ l.

The PCR condition: 94 ℃ of sex change 30s, IgG1, IgG3, κ, λ annealing temperature are respectively 62 ℃, 64 ℃, 60 ℃, 67.5 ℃, time 30s, 72 ℃ are extended 30s, totally 35 circulations, 72 ℃ of continuing extend 5min, 4 ℃ of terminations.Get 5 μ l PCR products after reaction finishes and carry out the agarose gel electrophoresis detection.

5, the pComb3Hss plasmid is gone in light chain κ, λ gene clone, get the L-pComb3Hss plasmid, make up the light chain library.The structure iron of pComb3Hss plasmid is as shown in Figure 2.

The pComb3Hss DNA is carried out the SacI/XbaI double digestion, and glue reclaims purpose segment DNA, and the carrier dephosphorylation is handled.The κ, the lambda light chain gene PCR product that obtain are carried out purifying, and the κ/lambda light chain gene PCR product that then purifying is obtained carries out the SacI/XbaI double digestion, at last carrier is connected with target gene fragment.To connect product L-pComb3Hss and transform the XL1-Blue competence bacteria, be coated with the dull and stereotyped storage capacity of measuring, and increase bacterium and cultivate, extracting light chain reorganization phagemid.SacI/XbaI double digestion and XhoI single endonuclease digestion are identified.

6, heavy chain Fd gene is written into the L-pComb3Hss plasmid, H+L-pComb3Hss (below be abbreviated as H+L+P) plasmid, make up and accomplish combinatorial library.

The L+P DNA is carried out 37 ℃ of water-bath enzymes of XhoI/SpeI double digestion to be cut 2 hours.Behind 1% agarose gel electrophoresis, glue reclaims the DNA band of 4054bp, and carries out dephosphorylation and handle.Behind the G1/G3 heavy chain Fd gene PCR product purification that obtains, use the XhoI/SpeI double digestion, 37 ℃ of water-bath enzymes are cut 8h, at last carrier are connected with target gene fragment.To connect product H+L-pComb3Hss and transform XL 1-Blue competence bacteria, be coated with the dull and stereotyped storage capacity of measuring.

7, accomplish phage antibody library

With recombinant plasmid H+L+P transformed into escherichia coli XL1-Blue, infect with helper phage M13KO7.The XL1-Blue that will contain reorganization phagemid H+L+P shakes the bacterium enlarged culturing, adds 10 behind the 2h ¹²The helper phage M13 KO7 of pfu continues vibration 2h, adds kantlex (final concentration is 70 μ l/ml), 30 ℃ of overnight cultures.Centrifugal collection supernatant adds PEG8000 and NaCl (final concentration is respectively 4% and 3%), ice bath 30min; 15000r/m under 4 ℃ of conditions, centrifugal 10min abandons supernatant; Resolution of precipitate contains in the PBS liquid of 1%BSA in 2ml; 15000r/m under 4 ℃ of conditions, centrifugal 2min, collecting supernatant is antinuclear antibody Fab phage antibody library.Antibody library solution and XL1-Blue mixing after the dilution are coated with flat board, measure phage antibody library storage capacity.

(2) the segmental screening of humanized's antinuclear antibody Fab

1, the affine enrichment of antinuclear antibody Fab phage antibody

(1) get commercialization anti-dsDNA ELISA detection kit microwell plate, every hole adds the antinuclear antibody Fab phage antibody library stoste of 100 μ l, hatches 2 hours for 37 ℃.

(2) phage antibody library in the microwell plate is discarded, wash each 1 minute 10 times with the PBS liquid that contains 0.05%Tween-20.

(3) adding 50 μ l elutriants (0.1mol/L HCl, glycocoll adjust pH to 2.2 add BSA to final concentration 0.1%) room temperature left standstill 10 minutes.

(4), add the Tris neutralization of 2mol/L immediately with elutriant piping and druming back sucking-off.

(5) the XL1-Blue intestinal bacteria nutrient solution of adding prepared fresh is after room temperature leaves standstill 15min.

(6) add tsiklomitsin and penbritin.

(7) get 1 μ l coated plate and measure the phage elution amount, change LB (Amp+Tet) 100ml behind all the other 37 ℃ of shaking culture 1h over to,

(8) press 10 behind the 1h ¹²The final concentration of pfu/ml adds helper phage M13KO7.

(9) adding kantlex to final concentration behind the vibration 2h is 70ng/ml, 30 ℃ of overnight cultures.

(10) next day centrifugal, supernatant is with PEG and NaCl deposition, redissolves with the PBS that contains 1%BSA more promptly to obtain secondary phage antibody library.By the said mensuration of carrying out the phage antibody library titre of 1.2.2.

(11) the said method of 1-10 repeats 4 and takes turns screening set by step.

2, the mensuration of phage antibody library titre

(1) phage antibody library stoste or secondary antibodies storehouse solution are done 10 times with the LB nutrient solution and are diluted to 10 under aseptic condition ^-4, 10 ^-5, 10 ^-6, 10 ^-7, 10 ^-8The antibody library solution for standby.

(2) get single intestinal bacteria XL1-Blue bacterium colony, contain in the LB nutrient solution of tsiklomitsin 37 ℃ at 2ml and spend the night.

(3) change kind with 2% and contain in the LB nutrient solution of tsiklomitsin, be expanded to A in 2ml ₆₀₀Value is 0.2～0.3 o'clock, and per 100 μ lXL1-Blue bacterium liquid add the phage antibody library solution after the 1 μ l dilution, and metainfective XL1-Blue evenly coats in LB (Amp+Tet) agar plate, is inverted in 37 ℃ of incubators after doing slightly to absorb, and cultivates 10h.

(4) the dull and stereotyped colony count of statistics LB calculates the phage antibody library titre.All the other 3 are taken turns screening and measure the phage antibody library titre with method.

3, the preliminary evaluation of phage antibody

(1) with 4 take turns wash-out after the enrichment phage antibody library infect XL1-Blue.

(2) bed board.

(3) picking 90 strain mono-clonal bacterium colonies are inoculated into 2ml respectively and contain in the LB nutrient solution of tsiklomitsin (10 μ g/ml) and Pyocianil (20 μ g/ml).

(4) 37 ℃ of shaking culture pressed 10 after 1 hour ¹²The final concentration of pfu/ml adds helper phage M13KO7.

(5) continue to cultivate that to add kantlex to final concentration after 2 hours be 70 μ g/ml, 30 ℃ of overnight cultures.

(6) next day centrifugal after, respectively get of the PBS dilution that contain 1%BSA of 50 μ l supernatants with equivalent, join respectively in the anti-dsDNAELISA detection kit microwell plate, hatched 2 hours.

(7) each hole liquid is removed in suction, washes 4 times with the PBS liquid that contains 0.05%Tween-20.

(8) add HRP-goat anti-human igg Fab antibody.

(9) reaction is inhaled after 1 hour and is removed each hole liquid, and PBS liquid is washed 4 times.

(10) add TMB/H ₂O ₂Chromogenic substrate, lucifuge, termination reaction after 30 minutes is measured A ₄₅₀Value.

4, antinuclear antibody Fab is segmental obtains

(1) selects A in the phage antibody evaluation ₄₅₀Be worth the single bacterium colony of 2 the highest strains, amplification back extracting phagemid

(2) extractive phagemid is carried out the SpeI/NheI double digestion, reaction system is following:

37 ℃ of water-bath enzymes are cut 2h, 1% agarose gel electrophoresis.

Behind (3) 1% agarose gel electrophoresis, glue reclaims the DNA band of 4176bp, and concrete steps are following:

1. cut off the sepharose that contains target DNA fragment, put into the 1.5ml centrifuge tube.

2. add the ratio adding S1 liquid of 300 μ l S1 liquid in every 100mg sepharose, put 50 ℃ of water-baths 10 minutes, sepharose is dissolved fully.Put upside down mixing once in per 2 minutes.

The gel liquid that 3. will dissolve moves into adsorption column, centrifugal 30 seconds.Outwell liquid in the collection tube, again adsorption column is put into collection tube.

4. in adsorption column, add 500 μ l W1 liquid, centrifugal 15 seconds.Outwell liquid in the collection tube, adsorption column is put into collection tube.

5. in adsorption column, add 500 μ l W1 liquid, leave standstill 1 minute after, centrifugal 15 seconds.Outwell liquid in the collection tube, adsorption column is put into collection tube.

6. centrifugal 1 minute.

7. adsorption column is put into clean 1.5ml centrifuge tube, central authorities add 30 μ l T1 liquid at adsorption film, leave standstill 1 minute after, centrifugal 1 minute, as early as possible the purpose fragment that obtains is carried out next step experiment.

(4) dna fragmentation that glue is reclaimed (removing the H+L+P fragment of gIII gene) carries out from connecting.Reaction system is following:

Add ddH ₂O to 10 μ l, 16 ℃ of water-baths are spent the night

(5) get part from the DNA that connects, row 1% agarose gel electrophoresis behind the Xho I single endonuclease digestion connects effect to identify.

Reaction system is following:

37 ℃ of water-bath enzymes are cut 2h, 1% agarose gel electrophoresis.

(6) confirm that the DNA that will connect certainly after the successful connection is converted into XL1-Blue, 3 single bacterium colony amplifications of each picking, 37 ℃ are cultured to A ₆₀₀Be that 0.5 o'clock adding inductor IPTG is 1mmol/L to final concentration, 30 ℃ of shaken overnight.

(7) collect thalline, behind the multigelation 4 ℃ centrifugal, preserve supernatants for-20 ℃, purpose antibody, i.e. humanized's monoclonal anti antinuclear antibodies.

The phage antibody library that adopts normal people's repertoire antibody variable region gene to make up can comprise various antibody theoretically; But for a part of antibody, lack enough abundance; Be unfavorable for further enrichment screening, require a great deal of time and possibility of success very low.Antinuclear antibody (ANA) abundance in normal population is extremely low, and the difficulty that from normal people or mile abnormality Immunological diseases patient's antibody variable gene, extract the ANA gene is bigger.The antinuclear antibody Fab phage antibody library gene source that this experiment makes up helps obtaining abundant antinuclear antibody expressing gene in autoimmunization patient's peripheral blood lymphocyte of ANA IIF detection titre greater than 1: 10000; Simultaneously; Consider that ANA target antigen structural form difference is bigger; For guaranteeing the multifarious existence of ANA and further improve ANA gene abundance that the present invention has separated several satisfactory autoimmunization patient's peripheral blood lymphocytes and made up phage antibody library jointly.This experiment identifies that to connecting product the connection product of selecting qualified batch transforms, and is intended to reduce invalid storage capacity before plasmid transforms.Experimental result shows: to 3 bacterium colonies of 4 bacterium colonies of ANA Fab antibody library and ANA Fab phage antibody library all confirm to comprise goal gene in the recombinant products with machine testing, show that the validity of storage capacity is good.What this experiment was adopted is that conventional Calcium Chloride Method transforms phagemid, simple, but the too late electrotransformation of transformation efficiency, through preparing super competence intestinal bacteria XL1-Blue, experimental result shows that storage capacity is 10 ⁴The order of magnitude, the high abundance property and the good validity of storage capacity of consideration antibody library gene source, this storage capacity can satisfy the screening of next step high-affinity antibody.In a word; Autoimmunization patient's peripheral blood lymphocyte of selecting the high titre of ANA is as the antibody library gene source; Help making up a relative high abundance, low storage capacity; High efficiency humanized ANA IgF ab fragment phage antibody library, thus be convenient to filter out satisfactory high-affinity antibody.

The present invention guarantees that through following measure the enzyme of PCR product cuts effect, smoothly construction recombination plasmid.

Add the proper protection base when 1, designing primer, conscientiously check the base sequence of the synthetic primer and the synthetic primer of submitting to.

2, guarantee the activity of used restriction endonuclease.

The amount of all restriction endonucleases when 3, the increasing enzyme is cut, the proper extension enzyme is cut the time.

4, the pcr amplification condition of strict goal gene with sepharose confirmatory experiment effect, and is carried out purifying and recovering to the PCR product to the PCR product, does not use the long purifying fragment of period of storage to be used for enzyme as far as possible and cuts and be connected.

Sequence table

 

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Claims (3)

1. the preparation method of humanized's antinuclear antibody is characterized in that may further comprise the steps:

(a) collect the autoimmune disease peripheral blood mononuclear cells, extracted total RNA, the cDNA library is obtained in reverse transcription;

(b) be masterplate with the cDNA library that obtains, PCR method amplification light chain k, l gene and immunoglobulin molecules heavy chain Fd gene;

(c) the pComb3Hss carrier is gone in light chain k, l gene clone, make up the light chain library;

(d) heavy chain Fd gene is written into step c gained carrier, makes up and accomplish combinatorial library;

(e) with steps d gained recombinant plasmid transformed intestinal bacteria XL1-Blue, infect with helper phage M13KO7, combinatorial library at random is expressed in the filobactivirus surface, accomplishes phage display;

(f) take turns naughty sieve through 4; Humanized's antinuclear antibody Fab phage antibody library that enrichment has made up; Indirect elisa method identifies that 4 take turns naughty sieve back antinuclear antibody Fab phage antibody, extracts the phagemid dna of positive colony, excision gIII gene fragment; From connecting back transformed into escherichia coli XL1-Blue, with IPTG abduction delivering solubility humanized antinuclear antibody Fab fragment; Using indirect elisa method and fluorescent immune method identifies expression product;

(g) utilize affinity chromatography column purification humanized antinuclear antibody Fab fragment.

2. preparation method as claimed in claim 1 is characterized in that among the step a, the titre of antinuclear antibody is greater than 1:10000 in autoimmunization patient's blood.

3. preparation method as claimed in claim 1 is characterized in that among the step b, and the primer sequence that the amplification of PCR method is used is shown in SEQ ID No.1 ~ SEQ ID No.8:

Variable region of heavy chain 5 ' end primer VH4f:

5′-CAGGTGCAGCTGCTCGAGTCGGG-3′；（SEQ?ID?No.1）

Variable region of heavy chain 5 ' end primer VH1a:

5′-CAGGTGCAGCTCGAGCAGTCTGGG-3′；（SEQ?ID?No.2）

Heavy chain IgG1 (Fd) 3 ' end primer CG1Z:

5′-GCATGTACTAGTTTTGTCCCAAGATTTGGG-3′；（SEQ?ID?No.3）

Heavy chain IgG3 (Fd) 3 ' end primer CG3Z:

5′-TGTGTGACTAGTGTCACCAAGTGGGGTTTT-3′；（SEQ?ID?No.4）

Light chain (k) variable region 5 ' end primer Vk3a:

5′-GAAATTGAGCTCACGCAGTCTCCA-3′；（SEQ?ID?No.5）

Light chain (l) variable region 5 ' end primer VL4:

5′-TCTGAAGAGCTCCAGGACCCTGTTGTGTCTGTG-3′；（SEQ?ID?No.6）

Light chain (k) constant region 3 ' end primer Ck1Z:

5′-GCGCCGTCTAGAAACACTCTCCTCTGTTGAAGCTCTTTGTGACGGATCTCAG-3′；（SEQ?ID?No.7）

Light chain (l) constant region 3 ' end primer CL2:

5′-CGCCGTCTAGAACTATGAACATTCTGTAGG-3′；（SEQ?ID?No.8）。

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