CN1442429A - Preparation method of human source anti-HBsFab - Google Patents

Abstract

A process for preparing the humanized anti-HBsFab includes such steps as respectively cloning the Fd and light-chain genes of HBs in pBAD, inserting pBAD-light chain compound in the carrier with cloned Fd, respectively expressing double promoters and dual peptides, and directional secretion and self assembly of somatic periplasm cavity. Its advantage is the obviously improved Fab expression and activity.

Description

The people source is anti--preparation method of HBsFab

Technical field

The present invention relates to biological technical field.Be specifically related to the people source anti--preparation method of HBsFab.

Background technology

Hepatitis B is the common communicable disease of China, does not still have the specific treatment means at present.Discover that it is active that anti-HBsAg antibody has protection, can make human body avoid the invasion and attack of hepatitis B virus.Applied Biotechnology prokaryotic expression people source is anti--and HBsFab comes from ZebedeeSL the earliest, after this has many places to prepare the report of anti-HBsFab of people source biotechnology and ScFv in succession both at home and abroad.Wang Zhiyi etc., Chinese hepatopathy magazine, 1999,7 (3); Wang Yan etc., Chinese microorganism and Journal of Immunology, 1995,15 (5); Gao Hui etc., Chinese Journal of Pathophysiology, the pericentral siphon solinocrine expression that is prokaryotic system of 2001,17 (4) reports.Up to now, the combinatorial antibody library technology is all adopted in the preparation of all anti-HBsFab, uses the pComb3 carrier, and the ITPG abduction delivering is in the experimental study stage always.

Summary of the invention

Technical problem to be solved by this invention is to express on the basis of anti-HBsFab at former pComb3, and the pComb3 expression system is converted to the pBAD system, provide a kind of economic, practical, batch preparations people source anti--preparation method of HBsFab.

The preparation method of the people disclosed by the invention anti-HBsFab in source adopts the Fd that clones anti-HBs respectively and light chain gene in pBAD, again pBAD-light chain complex body is inserted in the carrier of cloning Fd, formation double-promoter, two peptide are expressed respectively, and the directed secretion in the thalline pericentral siphon chamber mode of assembling voluntarily prepares the active antibody fragment.

Concrete preparation process comprises:

1, selecting the cloning vector of commercialization high-expression vector pBAD/gIII as this product, is template with the plasmid of pComb3/anti-HBsFab bacterial strain, designs following primer respectively according to the requirement and the Fab gene structure of pBAD carrier: Fd chain Forward; 5 '-TG AAG CTGCTC AGA TCT GGG, Backward; 5 '-TTA CTA AGA TCT TTT GTCACA AGA TTT GG, lambda light chain; Forward; 5 '-ATG GCC GAG CTC CAGCCT G, Backward; 5 '-TT AAC TCT AGA ACT ATG AAC.Fd and lambda light chain, electrophoresis and glue recovery purpose fragment increase respectively.

2, the specificity restriction enzyme site of being introduced according to Fd and lambda light chain two ends, with identical restriction endonuclease respectively enzyme cut carrier, electrophoresis and glue reclaim carrier segments, are connected with the lambda light chain fragment with corresponding Fd with ligase enzyme respectively, make up pBAHBs-Fd and pBAHBs-Lc single chain antibody fragments expression vector.

3, after the connection product transforms the Top10 bacterium, cut evaluation through PCR method or enzyme, screening purpose single chain antibody fragments gene masculine clone, the pBAD of formation starts single peptide expression pattern.

4, design special primer prepares the nucleotide sequence that comprises lambda light chain antibody fragment gene and promotor, homing sequence in the gene amplification mode.

The nucleotide sequence of the carrier that contains Fd antibody fragment gene that 5, double digestion has been cloned and step 4 amplification preparation, electrophoresis and glue reclaim the purpose fragment, connect with ligase enzyme, and the recombinant plasmid figure that changes behind the structure sees Fig. 7;

6, after carrier transforms the Top10 bacterium, cut evaluation through order-checking and enzyme, screening positive clone increases bacterium, the selected clone of fermentation culture, and expressed fusion protein under the pectinose inductive condition carries out double startup and expresses test, the feasibility that the two peptides of checking are expressed synchronously.

7, the screening positive strain carries out the segmental batch preparations of human antibody.

Secretion expression's carrier of the present invention is pBAD/gIII, and its structure is seen Fig. 6.

The preparation of the embodiment of the invention 1 is described the people source in detail anti--HBsFab.The order-checking of anti-HBsFab through the present invention is obtained, be the forward sequencing primer with 5 '-GTG CCG TTC TAT AGC CATAGC respectively, 5 '-ATT GTT ATT ACT CGC GGC CC is the forward sequencing primer, entrust Boxing Gene Chip Co., Ltd., Shanghai to check order, record the Fd sequence and see sequence 1 (Fd-DNA), λ V sequence is seen sequence 2 (λ V-DNA).Through showing that with BLAST (Basic local alignment search tool) comparison sequencing result and expected results are in full accord, confirm the correct clone of human antibody gene.

The antibody protein that the present invention is obtained carries out the biological activity evaluation:

1. anti--direct the point sample of HBs Fab 3 μ l that will prepare is made negative control with BSA and irrelevant expression supernatant on nitrocellulose filter.Wait to do the back and seal with 5% skim-milk, room temperature was spent the night in 2 hours or 4 ℃, washed 3 times, added and diluted to such an extent that the HRP mark gets goat anti-human igg Fab at 1: 1000, and room temperature reaction 1 hour washs 3 times, added DAB colour developing liquid, after 15 minutes, added 2M H ₂SO ₄Termination reaction, result show that institute picking clone's dot blot result is all positive, see Fig. 1, as seen anti-in the arrow place-and HBsFab combines with the specificity of enzyme labelled antibody, and and BSA and enzyme labelled antibody do not have color reaction, and the reference source resists-the correct assembling of HBsFab.

2. anti--capable 15%SDS-PAGE the electrophoresis of HBsFab that will prepare, electrophoresis is complete to be transferred to isolating protein band on the nitrocellulose filter with half-dried transfer printing.With 5% skim-milk sealing, room temperature was spent the night in 2 hours or 4 ℃, wash 3 times, adds dilute at 1: 1000 the goat anti-human igg Fab of HRP mark, room temperature reaction 1 hour wash 3 times, the adding DAB liquid that develops the color, after 15 minutes, adding 2M H ₂SO ₄Termination reaction, Western blot the results are shown in Figure 3, in the visible positive band in the about 47.5kd of molecular weight place, also the reference source anti--the correct assembling of HBsFab.

The antigen-binding activity of anti--HBsFab that the present invention is obtained is studied:

With HBsAg as part, HBsAg (mg/ml) 3 μ l are anti--the HBsFab point sample on nitrocellulose filter, wait to do the back and seal with 5% skim-milk, room temperature was spent the night in 2 hours or 4 ℃, washed 3 times, added the anti--HBsFab of preparation, room temperature reaction 1 hour, wash 3 times, add dilute at 1: 1000 the goat anti-human igg Fab of HRP mark, room temperature reaction 1 hour, wash 3 times, add DAB colour developing liquid, after 15 minutes, add 2M H ₂SO ₄Termination reaction, result show the people source anti--HbsFab has extraordinary antigen binding capacity, the result that is positive in the arrow place sees Fig. 4.

2. the avidity of the mouse monoclonal antibody of animal's antibody that obtains through immunization route according to a certain antigen and hybridoma technology preparation is all in a metastable level, itself and the principle that the antigen association reaction of corresponding biotechnology antibody has competition to suppress are measured the antigen-specific avidity of anti--HBsFab.On nitrocellulose filter, carry out different antigen concentrations (3 μ g, 0.3 μ g, 0.03 μ g), the different antibodies inhibition concentration (10 times, etc. doubly, 1/10 times to anti--HBsFab) antigen-antibody reaction, add dilute at 1: 1000 the goat anti-human igg Fab of HRP mark, room temperature reaction 1 hour washs 3 times, adds DAB colour developing liquid, after 15 minutes, add 2MH ₂SO ₄Termination reaction, observations.The result show the people source of this systems produce anti--HBsFab has extraordinary antigen-binding activity, sees Fig. 5.1) 3 groups Fab all is positive, 2) 3 groups 3 kinds of negative controls are all feminine gender, 3) anti--HBs Fab group is all the positive and can display density poor to the antigen of different concns, 4) the mouse monoclonal anti-HBsAg of 3 kinds of concentration gradients does not all play restraining effect, 5) 10 times of concentration goat-anti-HBsAg (lane 1) present certain competition retarding effect, and it is thin out to show as the colour developing of HBsAg point sample spot.

The antibody that the present invention is obtained carries out studies on acute toxicity:

3 of picked at random Bal b/c mouse are one group, (0.05-0.5mg/m2 converts out the consumption of mouse by the human-body biological preparation only only to carry out intraperitoneal injection with 500 μ g/ with anti--HBsFab 250 μ g/ of purifying, use with 100 times of superelevation amounts with 50 times respectively), repeat once next day.The result show the people source anti--HBsFab do not have acute toxic reaction.

The outstanding advantage of people source biotechnology antibody fragment is clinical application, so high reactivity, high expression level are key points, the expression system that only reaches above-mentioned effect just has actual value.Manyly studies show that the active requirement that to satisfy Fab with the directed expression of polypeptides in the pericentral siphon chamber of E.Coli., the purpose that double-promoter is expressed, directed secretion also can reach correct assembling, but do not have as yet so far as commodity production, proceed to the data of clinical application, the wherein optimization of expression system, make it more practical, more economical, it is crucial being more suitable for producing in batches.The more former pComb3 of the expression system that the present invention relates to has a clear superiority in, increase bacterium, the fermentation positive strain obtain desired result, cell density OD ₆₀₀Reach 48.3, protein yield 80mg/L, expression amount and the activity of Fab all are significantly improved, and economy also is better than pComb 3 expression systems greatly, has possessed the condition of batch preparations.

Description of drawingsFig. 1 Dot-blot enzyme of Fig. 2 pBAD-Fab plasmid as a result cuts evaluation

Wherein 1 for there not being the pBAD of insertion single endonuclease digestion, and 2 is the Fab-pBAD single endonuclease digestion, and 3 are

The Fab-pBAD double digestion, 4 for the Western blot behind molecule Marker Fig. 3 Fab purifying as a result Fig. 4 antigen-binding activity as a result Fig. 5 affinity of antibody measurement result Fig. 6 expression vector pBAD/gIII structure iron Fig. 7 change recombinant plasmid collection of illustrative plates behind the structure

Embodiment

The amplification of embodiment 11, Fd and lambda light chain

The pComb3/anti-HBsFab bacterial strain is that this laboratory is self-built.Requirement and Fab gene structure according to the pBAD carrier design following primer respectively: Fd chain Forward; 5 '-TG AAGCTG CTC AGATCT GGG, Backward; 5 ' TTA CTA AGA TCTTTTGTC ACA AGA TTT GG, lambda light chain; Forward; 5 ' ATG GCC GAG CTCCAG CCT G, Backward; 5 '-TT AAC TCT AGA ACT ATG AAC.Fd and lambda light chain increase respectively.2, the clone of pBAHBs-Fd and pBAHBs-Lc

Vector plasmid is divided into two groups, uses Xho I and Bgl II double digestion for one group, another group Pst I and Xba I double digestion, and 1% agarose electrophoresis reclaims carrier DNA.Fd PCR product and Lc PCR product be with the double digestion that is same as cloning vector, is connected with ligase enzyme with carrier respectively after purified.Connect product transformed competence colibacillus TOP10, get single bacterium colony and increase bacterium with LB, plasmid DNA prepares the same, and positive bacterium colony is inserted in the screening of restriction enzyme digestion and electrophoresis method.3, the structure of pBAHBs-Fab carrier

(1) this fragment of the segmental preparation of pBAD/Lc is meant pBAD promotor and Lc fragment, but not complete light chain expression vector, the purpose of preparation is to insert another one pBAD promotor and Lc fragment in Fd heavy chain carrier again, forms single carrier structure of double-promoter, two peptide orientation expressions.Increase bacterium pBAD-Fd and pBAD-Lc positive colony respectively, Mini-prep extracts plasmid DNA, with pBAD-Lc two ends primer (Forward:5 '-GAA TTC AAACCA ATT GTC CAT ATT GC, Backward:5 '-TT AAC TCT AGA ACTATG AAC) amplify the pBAD/Lc fragment.

(2) pBAHBsFab clone EcoR I and Xba I be sure to pBAHBs-Fd carrier and pBAD/Lc amplified production of enzyme respectively, glue reclaims enzyme and cuts product, connects back transformed competence colibacillus TOP10 bacterial strain with ligase enzyme, for dual insertion screening.4, the screening of positive strain and evaluation

Screen dual insertion bacterium colony, the pectinose abduction delivering, PAGE and Western blot identify the expression effect of anti--HBsFab.

The enzyme of cloning vector is cut evaluation and is got the conversion bacterium colony, LB increases bacterium and spends the night, conventional method prepares the carrier of the dual insertion of plasmid DNA .1.Not I single endonuclease digestion, cutting nothing insertion pBAD carrier with same enzyme is contrast, agarose electrophoresis confirms the correct insertion of Fd and pBAD/Lc gene respectively at the visible enzyme slitting of 5.7Kb and 4.1Kb place band; 2.Xho I and Xba I double digestion show respectively at the visible enzyme slitting of 1.6Kb and 4.1Kb place band with Marker.See Fig. 2.

(2) abduction delivering of positive colony is got above-mentioned qualification result for the correct clone who inserts, and increases bacterium to OD ₆₀₀1.4 the adding final concentration is 0.02% pectinose abduction delivering, 28-30C spends the night.Be equipped with bacterium liquid supernatant by the multigelation legal system next day, and 10% PAGE is through Coommasia blue dyeing colour developing, the expression product band that as seen concentrates in about 47.5Kd place.See Fig. 3.5, Expression of Fusion Protein and purifying

The people source is anti--and this chamber of pBAD/gIII expression system of HBs Fab is self-built.Choose the positive colony of screening, be inoculated in and (contain 50mg/ml ammonia benzyl) among the 2ml LB 37 ℃, 225rpm jolts and spends the night, transfer (containing 50mg/ml ammonia benzyl) 37 ℃ among the 100ml LB in 1% ratio, 225rpm jolts 4h, adds pectinose (final concentration is 0.02%) abduction delivering, the 4 ℃ of centrifugal receipts of 4500rpm * 20min bacterium behind the 6h, add 3-5ml precooling 1xPBS liquid by the wet bacterium of every gram, ultrasonic degradation, power 200-300W, 10 times, each 10 seconds, 10 seconds at interval, 4 ℃ of 4500rpm after the cracking, centrifugal 20min, collect supernatant, carry out affinitive layer purification with Ni-NTA Agarose post.

Express supernatant and combine 2 hours, dress post, 10 times of column volume washingss (300mmol/L NaCl, 20mmol/L imidazoles, 500mmol/L NaH in 4 ℃ with Ni-NTA Agarose ₂PO ₄, pH=8.0) washing column bed, elutriant (300mmol/L NaCl, 20mmol/L imidazoles, 500mmol/L NaH ₂PO ₄, pH=8.0) wash-out is collected elutriant, with Beckman Du640 nucleic acid and protein analyzer, measures protein content.

Choose the positive colony of screening, be inoculated in and (contain 50mg/ml ammonia benzyl) among the 10ml LB 37 ℃, 225rpm jolts and spends the night, transfer (containing 50mg/ml ammonia benzyl) 37 ℃ among the 100ml LB in 1% ratio, 225rpm jolts 4-5h, inserts fermentation culture 14h in the 2.5L KLF2000 fermentation system in 4% ratio, begins batch feeding in 4h, begin to add my sugared abduction delivering in 9h, centrifugal receipts bacterium behind the 14h is got a certain amount of thalline and adds 10 times of volume precooling 1xPBS liquid, ultrasonic degradation by 1: 10 (W/V), power 300-400W, 10 times, each 10 seconds, 10 seconds at interval, 5000rpm after the cracking, 4 ℃ of centrifugal 30min collect supernatant, carry out affinitive layer purification with the Ni-NTAAgarose post, through above-mentioned upper prop combination, washing, the wash-out several steps is collected elutriant, with Beckman Du640 nucleic acid and protein analyzer, measure protein content.

The detection of purified product

With 5 '-GTG CCG TTC TAT AGC CAT AGC is the forward sequencing primer, with 5 '-ATT GTT ATT ACT CGC GGC CC is the forward sequencing primer, entrust Boxing Gene Chip Co., Ltd., Shanghai to check order, record the Fd sequence and see sequence 1, record λ V sequence and see sequence 2.

Through showing that with BLAST (Basic local alignment search tool) comparison sequencing result and expected results are in full accord, confirm the correct clone of human antibody gene.

It is positive that institute picking clone's dot blot result all shows, and sees Fig. 1, and the reference source is anti--the correct assembling of HBsFab.

Western blot the results are shown in Figure 3, also the reference source anti--the correct assembling of HBsFab.The people source that obtains is anti--and the HbsFab molecular weight is about 47.5kd.

1 Fdtggagctcga gtctggggga ggctcggtac agccgggagg gtccctgaga ctctcctgtg 60cagcctctgg attcaccttt agcagctatg ccatgagttg ggtccgccag gctccaggga 120gggggctgga gtgggtctca ggtatcagtg gtagtggtgg cacaacatat tacgctgact 180ccgtgaaggg ccgcttcatc atctccagag acaactccaa gaacatggtg tatctccaaa 240tgaacaccct gagagccgag gacacggccc tatattactg tgtgaagaat gcggggcagc 300agtggcgctt tgacgactgg ggccagggaa ccctggtcac cgtctcttca gcctccacca 360agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg ggcacagcgg 420ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg tggaactcag 480gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca ggactctact 540ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc tacatctgca 600acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc aaatcttgtg 660acaaaaacta gccatcacca tcaccatcat tagtaacacg acaggtttcc cgactggaaa 7202 λVcgcctggccg agctccagcc tgcctccgtc tctgggtctc ctggacagtc gatcaccatc 60tcctgcactg gaaccagcag tgacgttggt gcttatgact ttgtctcctg gtaccaacaa 120cacccaggca aaccccccaa actcatcatt tttgatgtca agaagcggcc cccaggggtt 180tccaatcgct tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc 240caaactgagg acgaggctga ttattactgc agctcatata caaacaccgt cacccccgtt 300ttcggcggag agaccaaggt gaccgtccta ggtcagccca aggctgcccc ctcggtcact 360ctgttcccgc cctcctctga ggagcttcaa gccaacaagg ccacactggt gtgtctcata 420agtgacttct acccgggagc cgtgacagtg gcctggaagg cagatagcag ccccgtcaag 480gcgggagtgg agaccaccac accctccaaa caaagcaaca acaagtacgc ggccagcagc 540tatctgagcc tgacgcctga gcagtggaag tcccacagaa gctacagctg ccaggtcacg 600catgaaggga gcaccgtgga gaagacagtg gcccctacag aatgttcata gttctagaac 660aaaaactcat ctcagaagaa gatctgaata gcgccgtcga ccatcattat 710

Claims (4)

1, the people source anti--preparation method of HbsFab, it is characterized in that this method adopts the Fd that clones anti-HBs respectively and light chain gene in pBAD, again pBAD-light chain complex body is inserted in the carrier of cloning Fd, formation double-promoter, two peptide are expressed respectively, and the directed secretion in thalline pericentral siphon chamber is the mode of assembling voluntarily.

2, people according to claim 1 source anti--preparation method of HbsFab, it is characterized in that described preparation method comprises the following steps:

1) selecting the cloning vector of commercialization high-expression vector pBAD/gIII as this product, is template with the plasmid of pComb3/anti-HBsFab bacterial strain, designs following primer respectively according to the requirement and the Fab gene structure of pBAD carrier: Fd chain Forward; 5 '-TG AAG CTGCTC AGA TCT GGG, Backward; 5 '-TTA CTA AGA TCT TTT GTCACA AGA TTT GG lambda light chain; Forward; 5 '-ATG GCC GAG CTC CAGCCT G, Backward; 5 '-TT AAC TCT AGA ACT ATG AAC, increase respectively Fd and lambda light chain, electrophoresis and glue reclaim the purpose fragment;

2) the specificity restriction enzyme site of being introduced according to Fd and lambda light chain two ends, with identical restriction endonuclease respectively enzyme cut carrier, electrophoresis and glue reclaim carrier segments, are connected with the lambda light chain fragment with corresponding Fd with ligase enzyme respectively, make up pBAHBs-Fd and pBAHBs-Lc single chain antibody fragments expression vector;

3) after the connection product transforms the Top10 bacterium, cut evaluation through PCR method or enzyme, screening purpose single chain antibody fragments gene masculine clone, the pBAD of formation starts single peptide expression pattern;

4) design special primer prepares the nucleotide sequence that comprises lambda light chain antibody fragment gene and promotor, homing sequence in the gene amplification mode;

The nucleotide sequence of the carrier that contains Fd antibody fragment gene that 5) double digestion has been cloned and step 4 amplification preparation, electrophoresis and glue reclaim the purpose fragment, connect with ligase enzyme, and the recombinant plasmid figure that changes behind the structure sees Fig. 6;

6) after carrier transforms the Top10 bacterium, cut evaluation through order-checking and enzyme, screening positive clone increases bacterium, the selected clone of fermentation culture, and expressed fusion protein under the pectinose inductive condition carries out double startup and expresses test, the feasibility that the two peptides of checking are expressed synchronously;

7) the screening positive strain carries out the segmental batch preparations of human antibody.

3, people according to claim 1 source anti--preparation method of HbsFab, it is characterized in that being that wherein the Fd amplimer is Forward; 5 '-TG AAG CTG CTC AGA TCTGGG, Backward; 5 '-TTA CTA AGA TCT TTT GTC ACA AGA TTTGG.

4, people according to claim 1 source anti--preparation method of HbsFab, it is characterized in that being that wherein the lambda light chain amplimer is Forward; 5 '-ATG GCC GAG CTC CAGCCT G, Backward; 5 '-TT AAC TCT AGA ACT ATG AAC.

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