CN1280985A - Recombined human hopatocyte auxin, its preparing process and its clinical application - Google Patents
Recombined human hopatocyte auxin, its preparing process and its clinical application Download PDFInfo
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- CN1280985A CN1280985A CN 99110801 CN99110801A CN1280985A CN 1280985 A CN1280985 A CN 1280985A CN 99110801 CN99110801 CN 99110801 CN 99110801 A CN99110801 A CN 99110801A CN 1280985 A CN1280985 A CN 1280985A
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Abstract
A differential indication PCR technique is used to obtain a new complete gene able to promote the repair of damaged liver cells and with substance total length of 0.7kb by screening the CDNA library of human foetal liver. The efficient expression strain is constituted. The induction expression of engineering bacteria, the separation and cracking of inclusion body and the process for restoring and decontaminating protein are built up to obtain high-purity recombined human hepatocyte auxin. It can externally promote the reproduction of primary culture liver cells and liver cancer cells BEL-7402 and internally promote the synthesis of mouse liver cell DNA after CCL4 is damaged and the repair of liver cells. It may be used to treat serious hepatitis, chronic hepatitis, liver fibrosis and cirrhosis.
Description
The present invention relates to by the cDNA of difference pcr clone nucleotide sequence analysis and the method for producing reorganization biological products one recombination human source hepatocyte growth-promoting factors, the detection method of vivo and vitro biologic activity to the recombination human source hepatocyte growth-promoting factors.This expression of gene product has clear and definite using value in clinical liver disease.
After the big exhibitor tire of old one-tenth liver therapy for treating hepatitis gravis, the domestic upsurge that once started people's tire liver therapy in the eighties.Zhang Yijun develops on the basis of hepatocyte stimulatory substance (HSS) at Labreque, successfully utilize the sucking pig liver to develop hepatocyte growth-promoting factors (pHGF), see CN93120999.4 " hepatocyte growth-promoting factors ", organize units concerned's clinical verification through State Scientific and Technological Commission's " the Seventh Five-Year Plan ", " eight or five " heavy liver tackling key problem group, confirm that pHGF treats hepatitis gravis clinically, obtain satisfied curative effect.Exploration through surplus 10 years has proved that now pHGF can induce the BEL-7402 apoptosis, to using CCL
4The hepatic fibrosis of leading to has blocking-up or restraining effect.
Because pHGF is one group of polypeptide class mixture,, be difficult to further its mechanism of action of research in view of many difficulties of separation purification method.Therefore, the molecular cloning work of this factor all is devoted in the laboratory between many both at home and abroad.
The objective of the invention is to clone the cDNA of recombination human source hepatocyte growth-promoting factors, analyze its nucleotide sequence, produce the method for reorganization biological products-recombination human source hepatocyte growth-promoting factors, detect its vivo and vitro biologic activity.
To achieve these goals, utilization variance round pcr of the present invention screens coding and promotes the proteinic cDNA of liver cell DNA synthetic, and utilizes prokaryotic expression system to study the biologic activity of its product from people's tire liver cDNA library.
The technical solution used in the present invention is: get induction of labor with water bag fetal liver cell and normal adult human liver tissue in age in April, extract mRNA respectively, carry out DD-PCR relatively with different primers, finishing screen is chosen people's hepatocyte growth-promoting factors coding region cDNA (sequence sees Table 1), the aminoacid sequence that comes out of deriving sees Table 2, and the Theoretical Calculation molecular weight is 15KDa.By prokaryotic expression system-escherichia coli expression, finally be purified into recombinant human hepatocyte growth-promoting factors albumen, SDS-PAGE result shows conforming to of molecular weight and expection, activity research shows that it has CCL
4Due to mouse liver injury provide protection is arranged, thereby may be the same with hepatocyte growth-promoting factors, applying clinical treatment hepatitis gravis, diseases such as chronic hepatitis and liver cirrhosis.
In a word, the present invention not only discloses a kind of nucleotide sequence of people's hepatocyte growth-promoting factors, and production method is provided, comprise the engineering bacteria derivational expression method, inclusion body is purified, series of process such as protein purification, but also the physico-chemical property and the biologic activity detection method of its product are provided.
The corresponding antibodies of purified product of the present invention preparation can be used as a kind of test kit and is used for detecting, and this product is expected applying clinical treatment hepatitis gravis in addition, the liver injury due to the chronic hepatitis, hepatic fibrosis or liver cirrhosis and other reason.
Implementing method of the present invention is specific as follows:
One, the cDNA of people's hepatocyte growth-promoting factors clone
1.mRNA extraction: get people's tire hepatic tissue and normal adult liver group about 4 months
Knit each 200mg, press the mRNA of Boehisnger company separating kit and extract
MRNA, the denaturing formaldehyde gel electrophoresis shows that it is good that the mRNA integrity is put forward by institute.
2. design of primers
3’?-(T-rich)-primers：
A：5’?-GCG?CAA?GCT?TTT?TTT?TTT?TTC?T-3’
B：5’?-GCG?CAA?GCT?TTT?TTT?TTT?TTC?C-3’
C：5’?-GCG?CAA?GCT?TTT?TTT?TTT?TTC?G-3’
D：5’?-GCG?CAA?GCT?TTT?TTT?TTT?TTG?T-3’
E：5’?-GCG?CAA?GCT?TTT?TTT?TTT?TTG?G-3’
F：5’?-GCG?CAA?GCT?TTT?TTT?TTT?TTG?A-3’
G：5’?-GCG?CAA?GCT?TTT?TTT?TTT?TTA?T-3’
H：5’?-GCG?CAA?GCT?TTT?TTT?TTT?TTA?C-3’
I：5’?-GCG?CAA?GCT?TTT?TTT?TTT?TTA?G-3’
J：5’?-GCG?CAA?GCT?TTT?TTT?TTT?TTA?A-3’
K:5 '-GCG CAA GCT TTT TTT TTT TTC A-3 ' 5 '-random primer:
00：5’?-CGG?GAA?GCT?TAT?CGA?CTC?CAA?G-3’
01：5’?-CGG?GAA?GCT?TTA?GCT?AGC?ATG?G-3’
02：5’?-CGG?GAA?GCT?TGC?TAA?GAC?TAG?C-3’
03：5’?-CGG?GAA?GCT?TTG?CAG?TGT?GTG?A-3’
04：5’?-CGG?GAA?GCT?TGT?GAC?CAT?TGC?A-3’
05：5’?-CGG?GAA?GCT?TGT?CTG?CTA?GGT?A-2’
06：5’?-CGG?GAA?GCT?TGC?ATG?GTA?GTC?T-3’
07：5’?-CGG?GAA?GCT?TGT?GTT?GCA?CCA?T-3’
08：5’?-CGG?GAA?GCT?TAG?ACG?CTA?GTG?T-3’
09：5’?-CGG?GAA?GCT?TTA?GCT?AGC?AGA?C-3’
10：5’?-CGG?GAA?GCT?TCA?TGA?TGC?TAC?C-3’
11：5’?-CGG?GAA?GCT?TAC?TCC?ATG?ACT?C-3’
12：5’?-CGG?GAA?GCT?TAT?TAC?AAC?GAG?G-3’
13：5’?-CGG?GAA?GCT?TAT?TGG?ATT?GGT?C-3’
14：5’?-CGG?GAA?GCT?TAT?CTT?TCT?ACC?C-3’
15：5’?-CGG?GAA?GCT?TAT?TTT?TGG?CTC?C-3’
16：5’?-CGG?GAA?GCT?TTA?TCG?ATA?CAG?G-3’
17：5’?-CGG?GAA?GCT?TTA?TGG?TAA?AGG?G-3’
Synthesizing of article one chain of 18:5 '-CGG GAA GCT TTA TCG GTC ATA G-3 ' 3.cDNA
Get the mRNA 0.2 μ g of tire liver and adult liver respectively, add 3 ' end primer, 1.6 μ g, in 65 ℃ of insulation 10min, add mixing in the Boehringer company first chain cDNA synthetic system rapidly, after room temperature is placed 10min,, carry out reverse transcription reaction in 42 ℃ of insulation 60min, finish the back in 99 ℃, the 10min deactivation.
4.PCR amplification:
Getting the above-mentioned reactant of 1 μ l respectively is template, 10 * PCR damping fluid (500mM KCl, 100mM Tris.cl pH8.3,20mM MgCl
2), the 0.1mM dNTP of 1.5 μ l, the 20 μ M of 1.2 μ l be 3 ', 5 ' end primer of combination of the same race not, 5 times of dilutions of 1 μ l [α-
32P] dATP (800 μ Ci/uM, NEN Dupont), the Taq enzyme of 0.5 μ l, the DEPC water of 12.75 μ l.4 circulations of reaction earlier (94 ℃, 45 seconds; 41 ℃, 60 seconds; 72 ℃, 160 seconds), press down 18 circulations (94 ℃, 45 seconds; 41 ℃, 60 seconds; 72 ℃, 120 seconds).Reaction product 6% poly-propionic acid amide glue contains 7M urea.Dried glue, compressing tablet spends the night with Kodak XAR5 film.
5. the separation of differential gene
Press the difference band on the film, on gel, scale off and put into 1ml TE damping fluid, leave standstill 15 minutes after, get supernatant, add 1ml TE (containing 100mM Nacl) and boil 10min, room temperature, spend the night, get 5 μ l and do following PCR reaction with preparation probe, 5 μ l, 10 * PCR buffer, 2.5 μ l 1mM dNTP, 3 μ l 20uM5 '-primer, 3 μ l, 20 μ M, 3 '-primer, 1 μ l Taq enzyme, 5 μ l samples, DEPC water 30.5 μ l.94 ℃, 45S; 60 ℃, 60S; 72 ℃, 60S; Totally 25 circulations.Utilize the preparation of low melting point glue to reclaim probe.
6. utilize antibody and oligonucleotide screening people tire liver cDNA expression library:
Be built into people's tire liver cDNA library of lambda particles phage with the antiserum(antisera) screening of anti-hepatocyte growth-promoting factors.Obtain behind the positive colony again oligonucleotide probe screening with above-mentioned preparation.Above method is all by " molecular cloning experiment guide " second edition, Science Press, 1989.
7. the sequential analysis of positive colony
Positive colony is through the inscribe enzymatic analysis, and the external source that contains 0.7kb is inserted fragment, this external source is inserted fragment insert the pGEM-T plasmid, E.coli JM109 is experienced in conversion, the picking hickie is with CsCl density gradient centrifugation plasmid purification, as the T of template with Promega company
7The archaeal dna polymerase assay kit adopts two strands not hold cessation method, and product is analyzed through the genomy x of Beckman company dna sequencing instrument, reads nucleotide sequence after radioautograph, sees attached list 1.
Two, the prokaryotic expression of recombinant human hepatocyte growth-promoting factors and production
1. the structure of the foundation-engineering bacteria of coli expression system and expression
Press table 1 sequence synthesized primer thing
Primer 1:5 '-CGA ATT CAT GCG GAC GCA GCA GAAG-3 '
Primer 2: 5 '-CGG GAT CCC TAG TCA CAG GAG CCATC-3 '
With the positive colony is template, contains 1.5mmol/LMgCl in the 50ul reaction system
2, dNTPs O.1mmol/L, 0.2umol/L P1, the P2 primer, 10mmol/LTris HCL, 1.25u Taq archaeal dna polymerase, reaction parameter is: 95 ℃, 1min; 52 ℃, 1min; 72 ℃, 1min; After 35 circulations, extend 1Omin in 72 ℃, with 1.5% agarose electrophoresis, and the specific amplification fragment of recovery 384bp, through EcoRI, the prokaryotic expression carrier Pbv-220 that the BamHI enzyme is cut behind the 1hr with corresponding enzyme is cut is connected, and transformed competence colibacillus E.coli JM 109 paves plate (the LB plate that contains AMP) and spends the night for 30 ℃.Whether picking clone identifies positive clone through restriction analysis.After positive monoclonal inoculated 30 ℃ of sub-5ml LB (AMP 100 μ g/ML) nutrient solutions and spend the night, when inoculating the LB culture medium culturing to OD600=0.5 by 3%, improve rapidly culture temperature to 42 ℃, continue to cultivate 6 hours, centrifugal collection thalline checks that through SDS-PAGE showing has special 15KD unconventionality expression band, consistent with theoretical value calculating, expression product has promotion CCL
4Due to the recovery of mouse liver injury.
2. engineering bacteria high density fermentation and expression:
With 20% concentration seed liquor is inoculated in 30 ℃ of 8L LB nutrient solutions (containing AMP100ug/ml), 300 rev/mins of cultivations, carry out logical being measured of feed supplement (by 20% yeast powder) according to the bacterial growth state simultaneously and be 4L/min, dissolved oxygen 65%, stirring velocity is controlled at 300-700 rev/min, when treating OD600=2.0, be warming up to 42 ℃, abduction delivering 3.5-4.0 hour.Centrifugal collection bacterium, electrophoresis is identified the target protein expression amount.Wet bacterium harvest yield is 50g/L, and the target protein expression amount is 28-32%.
3. the cracking of bacterial cell
Centrifugal 20 minutes of 6000rpm, supernatant discarded adds lysis buffer (50mmol/L Tris HCL by 10% of wet bacterium weight, pH8.0,1mmol/L EDTA)-70 ℃, frozen spending the night, freeze thawing next day, ice-bath ultrasonic 30 seconds, 2min at interval, totally 8 times, behind ultrasonic the finishing, inspection bacterial cell disruption rate is more than 98%, 12000rpm * 20min, 4 ℃, abandon supernatant, collect inclusion body.
4. the washing of inclusion body
(1) with 50mmol/L Tris.cl, pH8.0,1mmol/l EDTA is resuspended
Precipitation, 120000rpm * 20 min abandons supernatant.
(2) with 0.5%Tirton X-100,2mmol/L beta-mercaptoethanol, 10mM/L
EDTA, 20mmol/l Tris.cl pH8.0 washing, 12000 rpm * 20
Min abandons supernatant, precipitation 20mmol/L Tris.cl pH8.0,1mmol/L
Supernatant is abandoned in the EDTA washing.
(3) with the 3M urea, 20mmol/L Tris.cl 2mmol/L EDTA washing,
The centrifugal supernatant of abandoning is weighed.
5. sex change, renaturation
The inclusion body of purifying is dissolved in the 8M urea by wet 1%, and in the 50mmol/L Tris.clpH8.0 damping fluid, 37 ℃, 30min is transparent to solution, limpid centrifugal 12000 rpm * 30 min, and supernatant is limpid transparent, for faint yellow.
The urea concentration of sex change liquid is diluted to 1M with 50mmol/L Tris.cl pH8.0, and this moment, solution should be limpid transparent, little yellow.
6. the bolt chromatography purification of recombinant protein
Select DEAE-Sepharose FF (Pharmacia) anion-exchange column for use, with 50mmol/L Tris.cl pH8.0 balance, renaturation solution is directly gone up sample earlier, and flow velocity is pressed 60ml/min.The same 50mmol/L Tris.cl pH8.0 wash-out of using is not in conjunction with phase, and with 300mmol/L NaCL stepwise elution, 280nm detects the eluted protein peak.The target protein peak is at first peak, and SDS-PAGE identifies that purity of protein is about 95%, the first peak sample is fully dialysed with 50mmol/L Tris.cl pH8.0 go up sample Sephacryl S-100 after Nacl is removed in the back, collects main peak, and target protein purity is greater than 98%.Three, recombinant human hepatocyte growth-promoting factors anti-liver injury activity and Monoclonal Antibody
1. activity in vivo detects
1.1 animal BALB/C mice body weight 18-20g male and female half and half are raised 1 and were supported for 1 week under the quiet condition,, inject once the 2nd damage grouping in back 24 hours, 10 every group at interval after 2 days again with 50%CCL4 whiteruss abdominal injection 1.5ml/kg body weight.(1) saline control group, 0.5ml/ mouse every day (2) pHGF group: every day 5mg/ mouse; (3) recombinant human hepatocyte growth-promoting factors (rhpHGF) purity＞95%, every day 10ug/ mouse.Treatment was killed mouse after 3 days continuously, got liver and did histological examination.The result shows that the saline control group has significant balloon sample and becomes, and nucleus is dense to be dyed, and circumference is unclear.Positive controls pHGF and rhpHGF group recover normal basically, have only a few cell swelling.Show the recombinant human hepatocyte growth-promoting factors the same with pHGF have the impatient liver damage of mouse experiment due to the CCL4 is had provide protection.
1.2 (the abdominal injection D-Gal (2g/kg body weight) of 250g ± 10g) was raised with granulated feed, 5% G/W, sub-cage rearing after 5 hours with male SD rat.42 of SD rats with survival after 48 hours are divided into 2 groups, and the disposable injection rhpHGF in treatment group abdominal cavity (400 μ g/kg body weight), control group inject with amount injection physiological saline.As a result, survival rate after 30 days: treatment group 39%, and control group only is 18%.Show and have the depleted effect of rats'liver due to the treatment D-Gal under the rhpHGF.
2. external activity detects
Get BEL-7402 liver cancer cell suspension (1 * 10
4/ ml) be inoculated in 96 porocyte culture plates, to contain 5% calf serum RPMI-1640 in 37 ℃, 5%CO
2Cultivate under the condition after 24 hours, add testing sample, every sample 4 holes are continued to cultivate and were surveyed MTT in 48 hours, and the result shows that the recombinant human hepatocyte growth-promoting factors has the effect of stimulating proliferation to 7402.
3. MONOCLONAL ANTIBODIES SPECIFIC FOR
3.1 immunity
RhpHGF is mixed with complete Freund's adjuvant, and it is subcutaneous to be injected in the female mouse inguinal region of BALB/C, injects once with the position after two weeks again, detects anti--rhpHGF antibody titers in the mouse group in back 40 days in injecting for the first time.
3.2 merge
Splenocyte and the SP2/0 myeloma cell of immunity success mouse are carried out cytogamy with 1: 5 ratio under the polyoxyethylene glycol condition.
3.3 clone
, should clone for the second time the back positive rate and reach 100% hybridoma cell clone with limiting dilution liquid, again behind row two time clonings, that hybridoma is frozen or carry out characteristic research.According to the clone result, there are 6 strains to secrete the monoclonal antibody of anti-rhpHGF.
Table 1
caacatggcggcgcccggcgagcggggccgcttccacggcgggaacctcttcttcctgccggggggcgcgcgctccgagatgatggacgacctggcgaccgacgcgcggggccggggcgcggggcggagagacgcggccgcctcggcctcgacgccagcccaggcgccgacctccgattctcctgtcgccgaggacgcctcccggaggcggccgtgccgggcctgcgtcgacttcaagacgtgg ATG CGG ACG CAG CAGAAG CGG GAC ACC AAG TTT AGG GAG GAC TGC CCG CCG GAT CGCGAG GAA CTG GGC CGC CAC AGC TGG GCT GTC CTC CAC ACC CTGGCC GCC TAC TAC CCC GAC CTG CCC ACC CCA GAA CAG CAG CAAGAC ATG GCC CAG TTC ATC CAT TTA TTT TCT AAG TTT TAC CCCTGT GAG GAG TGT GCT GAA GAC CTA AGA AAA AGG TTG TGC AGGAAC CAC CCA GAC ACC CGC ACC CGG GCA TGC TTC ACA CAG TGGCTG TGC CAC CTG CAC AAT GAA GTG AAC CGC AAG CTG GGC AAGCCT GAC TTC GAC TGC TCA AAA GTG GAT GAG CGC TGG CGC GACGGC TGG AAG GAT GGC TCC TGT GACTAGagggtggtcagccagagctcatgggacagctagccaggcatggttgcataggggcagggcactcattaaagtgcatcacagccagaaaaa2MRTQQ KRDTK FREDC PPDRE ELGRH 25SWAVL HTLAA YYPDL PTPEQ QQDMA 50QFIHL FSKFY PCEEC AEDLR KRLCR 75NHPDT RTRAC FTQWL CHLHN EVNRK 100LGKPD FDCSK VDERW RDGWK DGSCD 125
Claims (12)
1. one kind promotes to damage the material---people hepatocyte growth-promoting factors that liver cell is repaired, it is characterized in that containing a kind of nucleotide sequence of the people's of coding hepatocyte growth-promoting factors, and can encode recombination human source hepatocyte growth-promoting factors and other animal, as gene order and the dna artificial sequence synthetic of large and small mouse, pig, dog, rabbit, ox, sheep and primates.
2. according to the described nucleotide sequence of claim 1, it is characterized in that comprising the complementary strand of dna sequence dna.
3. according to the described nucleotide sequence of claim 1, it is characterized in that having specific aminoacid sequence, molecular weight is 15Kda.
4. method of producing the recombination human source hepatocyte growth-promoting factors, comprise engineering bacterium fermentation, the separating and cracking of inclusion body, the biologic activity of renaturation and protein purification and expression product, it is characterized in that after the described dna sequence dna insertion of claim 2 appropriate carriers, transform protokaryon or eukaryotic host cell, under conditions suitable, cultivate transformant, obtain people source reorganization hepatocyte growth-promoting factors polypeptide.
5. method according to claim 4 is characterized in that suitable carriers is a prokaryotic expression carrier, and prokaryotic hosts is intestinal bacteria.
6. method according to claim 4 is characterized in that the engineering bacteria high density fermentation, and its best abduction delivering time is cell density OD600=1.2-1.5.
7. method according to claim 4 is characterized by inclusion body sex change and 0.05M Tris.cl, the 8M urea of pH8.0 preparation.
8. according to the described method of claim 4, it is characterized by renaturation solution component is 1M urea 50mM Tris.cl, and pH8.0 or dilution refolding solution, its component are 50mMTris.cl.pH8.0.
9. method according to claim 4 is characterized by and adopts the column chromatography purification activated protein, DEAE-Sepharose FF anion-exchange column, and elutriant: 50mMTris.cl pH8.0,300mM Nacl, the 1st peak is the target protein peak.
10. recombination human source hepatocyte growth-promoting according to claim 1 is characterized in that promoting that external the DNA of primary cultured hepatocyte and liver cancer cell is synthetic, can promote CCL in the body
4Cause that to decrease back mouse liver cell DNA synthetic, and the damage liver cell is had the promotion repair, blocking-up and restraining effect are arranged using the hepatic fibrosis that CCL4 leads to.
11., as biological products treatment hepatopathy, it is characterized in that: SD rats'liver depletion due to the treatment D-Gal, survival rate treatment group 39% according to the described recombination human source hepatocyte growth-promoting factors of claim 1; And control group only is 18%.
12., it is characterized in that can be used for the treatment of hepatocellular injury due to a variety of causes, the blocking-up hepatic fibrosis according to the described recombination human source hepatocyte growth-promoting factors of claim 1.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101062946B (en) * | 2007-05-22 | 2010-09-29 | 中国人民解放军第二军医大学东方肝胆外科医院 | High-active liver regeneration reinforced factor and usage thereof |
| CN101869701A (en) * | 2010-06-30 | 2010-10-27 | 杭州华津药业股份有限公司 | Hepatocyte growth-promoting factor enteric-coated capsule |
-
1999
- 1999-07-20 CN CNB991108019A patent/CN1182155C/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101062946B (en) * | 2007-05-22 | 2010-09-29 | 中国人民解放军第二军医大学东方肝胆外科医院 | High-active liver regeneration reinforced factor and usage thereof |
| CN101869701A (en) * | 2010-06-30 | 2010-10-27 | 杭州华津药业股份有限公司 | Hepatocyte growth-promoting factor enteric-coated capsule |
| CN101869701B (en) * | 2010-06-30 | 2013-03-20 | 杭州华津药业股份有限公司 | Hepatocyte growth-promoting factor enteric-coated capsule |
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