CN1158895A

CN1158895A - Human liver proliferating agent

Info

Publication number: CN1158895A
Application number: CN 96103203
Authority: CN
Inventors: 杨晓明; 贺福初; 邱兆华; 谢玲; 吴祖泽; 宫锋; 胡志远; 魏汉东; 何浩
Original assignee: Institute of Radiation Medicine of CAMMS
Current assignee: Beijing C&n International Sci Tech Co ltd; Institute of Radiation Medicine of CAMMS
Priority date: 1996-03-08
Filing date: 1996-03-08
Publication date: 1997-09-10
Anticipated expiration: 2016-03-08
Also published as: CN1060520C

Abstract

By means of function screening method, is separated a whole-length cDNA capable of encoding a new liver proliferation promoting matter, named liver proliferating agent, containing 375 bp and open code-reading frame capable of encoding 125 amino acid proteins. Theoretic calculation shows the molecular weight of protein of 15 KDa, consistant with the SDS-PAGE result under denaturation condition. The recombined liver proliferating agent produced with proacryon and eucaryon can promote liver cell proliferation of rat with 40% cut liver and raise the survival of rat with liver function failure, and this shows that the recombined liver proliferating agent will be probably used for curing serious hepatitis, chronic active hepatitis and other serious hepatosis.

Description

Human liver proliferating agent

The present invention relates to the separation of human liver proliferating agent cDNA, nucleotide sequence analysis and produce the method for its proteins encoded and this gene expression product of aspect such as expression product hepatocyte proliferation activity evaluation has the potential clinical value.

2/3 liver divides excision PH rat blood can promote the normal rat hepatocyte growth through intersecting to circulate, inspired by this, people begin behind the PH rat peripheral blood and excavate special liver propagation stimulating factor, and the final protein of therefrom isolating a kind of propagation of row cell cultured supernatant by force, be pHGF (Hepatocyte Growth Factor, HGF), but further studies show that: this factor is not to resemble to have liver specificity desired, also non-its main target cell LaBrecque of liver cell etc. has described the earliest and has been present in that the specific specificity hepatocyte-stimulating factor in the liver tissues of rats is hepatic stimulator substance (Hepatic Stimulator Substance behind the PH, HSS), subsequently, this material of many experiment confirms extensively is present in the Mammals neonatal liver, in young baby liver and the regenerating hepatic tissue, but each family's name differs, thereby seem very chaotic, from people's tire liver, this type of factor that new calves liver and sucking pig liver etc. are extracted is widespread use clinical treatment Severe Viral Hepatitis, and obtain better curative effect, but originated by it, the limitation of species variation and biochemical preparation itself, the scale operation of these products and the raising of quality are subjected to bigger restriction, therefore, domestic, outer many families are devoted to the molecular cloning of this type of factor.

The object of the present invention is to provide a kind of human liver proliferating agent.

Late nineteen eighties, we have taken the lead in carrying out the research of hepatocyte proliferation activity material in people's tire liver at home, outward, its biological activity is being furtherd investigate, really contained in reference's tire hepatic tissue on a kind of basis of the new hepatocyte proliferation activity factor, utilizing ultrafiltration, DEAE-Cellulose, FPLC-monoQ, HPLC etc. carry out purifying to this active factor, and its relative reactivity improves nearly 50,000 times, but because the sealing of this protein amino terminal, determined amino acid sequence is not succeeded.Generally speaking, this factor fundamental characteristics is: the about 1.4～2.0KDa of molecular weight, and heat-resisting to the proteolytic enzyme sensitivity, can promote hepatocyte growth, its biological activity has organ specificity, no species specificity.From people's tire liver, extract poly (A)+mRHA, can translate the activity of this factor, show that this factor is the liver cell gene expression product at the Africa xenopus ovocyte.The present invention utilizes the functional expression sieve method, screens the cDNA of the short liver proliferin matter of coding from people's tire liver cDNA library, and the biological activity of its expression product is studied.

Extract mRNA from 4 monthly age induction of labor with water bag people tire hepatic tissues, with the pcDNAI plasmid is carrier, construction expression type people tire liver cDNA library, after the library carried out arranged, by functional expression screening human liver proliferating agent full-length cDNA, nucleotide sequencing shows that this clone contains the single open reading frame of 375bp (sequence is seen Fig. 1), and deduced amino acid is seen Fig. 2, theoretical molecular is 15KDa, and SDS-PAGE result under the sex change condition conforms to purification of protein.By former should, the eukaryotic system expressed products has the activity that promotes hepatocyte growth and anti-liver injury, thereby might applying clinical treatment hepatitis gravis, diseases such as chronic active hepatitis.

Implementation method of the present invention is as follows:

Fig. 1 is nucleotide sequence figure of the present invention.

Fig. 2 is aminoacid sequence figure of the present invention.

Fig. 3 is implementation method figure of the present invention.

Fig. 4 is the structure iron of pcDNAI plasmid.

Fig. 5 is the arranged synoptic diagram in library.

1.mRNA extraction

In the former research, we confirm that specific activity is the highest in the 4 monthly age people tire hepatic tissues, and therefore selecting 4 monthly age people tire hepatic tissues is parent material, utilize total RNA to extract, and mRNA purification kit (Promcga company) extracts mRNA.It is good that the denaturing formaldehyde gel electrophoresis shows that the mRHA integrity is put forward by institute.

2.cDNA building of library

Get the 10ugmRNA that carries, with 5 '-AATTCGCGGCCGC-(T) 15 is primer, it is synthetic that the cDNA of Pharmacia company synthetic agent box carries out cDNA, (the pcDNAI plasmid is available from Clontech company, structure is seen Fig. 4), and then utilize the EcoRI manual splice, to synthesize the cDNA orientation and be reconstituted in the pcDNAI plasmid, transformed into escherichia coli JM109 obtains 107 transformants, chooses transformant at random, through EcoRI, NotI restriction analysis, 4/7 transformant contains external source and inserts fragment, and the about 1.5Kb of mean size shows that institute's library quality of building is good.

3. the arranged in library and expression screening

Get about 10 from constructed library ⁸The bacterial suspension of recon, be diluted in the LB nutrient solution that contains penbritin, tsiklomitsin, by about 50 recons clone of every hole 100 μ L be inoculated in 96 porocyte culture plates (each plate reserve the 12nd row and H capable, use for permutation matrix), behind the culture plate mark in 30 ℃ of overnight incubation, get the 10u1 bacterial suspension respectively by every row (row) and be inoculated in corresponding row or column emptying aperture and form capable pond (pool), row pond (pool) (see figure 4) next day from each hole, the library is aligned to 210 minor matrixs like this, contains 210 * 77 * 50 recons approximately.

The library is a unit with the plate after arranging and finishing, and gets the 10ul cell suspension from row pond and row pond, being inoculated in 20ml (contains penbritin LB nutrient solution, extracts plasmid respectively, get about 10ug plasmid DNA, with standard DEAE-dextran method transfection cos-7 cell, cell density 4 * 10 is pressed in preceding 1 day of transfection ⁵/ 60mm ware goes down to posterity and cultivates the cos-7 cell, cultivated 24 hours with the RPMI-1640 that contains 5% calf serum, DEAE-dextran method transfection cos-7 cell is undertaken by Promega company test kit substantially, so be contrast, simultaneously the pcDNAI plasmid transfection is gone into the cos-7 cell, after the transfection 4 hours, with fresh RPMI-1640 washed cell secondary gently, and add the RPMI-1640 that 3ml does not contain serum, in 37 ℃ of 5%CO ₂Cultivate after 72 hours under the condition, collect culture supernatant and cell, after the ultrasonication, 65 ℃ were heated centrifugal 20 minutes of 72000g 10 minutes, receive supernatant, divide the excision model to carry out activity with 1/3 liver and detect, the positive pond of activated plate row, column cross bore bacterium is containing coated plate on the penbritin flat board, chooses single clone and carries out arranged, repeat above-mentioned screening process, until obtaining monospecific polyclonal.

4, biological activity assay, The selection result and positive colony sequential analysis

We have selected one of model of generally acknowledging in the world---and 1/3 liver divides the excision model to observe the hepatocyte proliferation activity of expression product.Get 200 ± 10gWister big white mouse, press the method row 1/3 liver branch excision that Higgens and Anderson introduce, 4 hours abdominal injection expression products of postoperative 1ml establishes physiological saline and lotus empty plasmid control group simultaneously, every group of 5 animals were pressed 100uci/kg body weight abdominal injection in 20 hours ³H-TdR puts to death rat and gets hepatic tissue in fixed position after 2 hours, carry out DNA extraction by the method for introductions such as Morley and reach ⁸H-TdR mixes counting, and the result calculates with cpm/ug DNA.Activity unit represents with stimulation index, stimulation index=experimental group cpm/ control group cpm value.Have short liver proliferation activity through the capable pond of G of functional screening the 28th plate and 8 row pond expression products, through one taking turns screening and obtain single positive and clone again, its expression product has short liver proliferation activity (stimulation index=3.2) and has dosage one effect positive correlation.This clone is carried out nucleotide sequences with terminal cessation method measure, show the single open reading frame (Fig. 1) that contains 375bp, the 125AA that can encode, molecular weight are the protein (Fig. 2) of 15KDa.

5. prokaryotic expression is produced liver proliferating agent

Press Fig. 1 sequence synthesized primer thing:

Primer 1:5 '-CGGAATTCATGCGGACGCAGCAGAAGC-3 ';

Primer 2: 5 '-GCGGATCCCTAGTCACAGGAGCCATCC-3 ';

With positive colony cDNA is template, contains 5mM primer 1, primer 2 at 100ul, 50mMKCL, 10mMTris-HCL (PH9.0), 0.1%TritonX-100,1.5mMMgCL2,200mMdNTP, 2.0uTaq archaeal dna polymerase (Promega company) system is carried out PCR, its parameter is: 95 ℃ 1 minute, 58 ℃ 1 minute, 72 ℃ 1 minute, totally 35 circulations circulate for the last time back 72 ℃ and extended 7 minutes; The PCR product carries out electrophoresis on 1.2% agarose gel electrophoresis, and the about 390bp specific amplified fragment of recovery, after purified, with EcoRI, the BamHI enzyme is cut 4h, and be connected with the prokaryotic expression carrier pBV-220 that cuts with corresponding enzyme (being so kind as to give) by Chinese professor Hou Yunde of Key State Laboratories Virogene Engineering For Virogene Engineering, get about 10ng and connect product, transform the competence E.coliJM109 of Calcium Chloride Method preparation with the standard test process, converted product is containing 30 ℃ of overnight incubation on the penbritin LB flat board, choose transformant and contain the cultivation of penbritin LB nutrient solution, extract plasmid, with EcoRI in 10ml, the BamHI enzyme is cut, three strain clones contain the external source insertion fragment of 390bp, and terminal cessation method nucleotide sequencing result is with table 1, and direction of insertion is correct; Single positive transformant is contained penbritin LB nutrient solution in 10ml cultivated liquid for 30 ℃, next day by 2% in 30 ℃ of amplification cultivation of LB nutrient solution to 0D600 ≈ 0.4～0.5, improve rapidly culture temperature to 42 ℃, continue to cultivate 5 hours, centrifugal collection bacterium, SDS-PAGE detects, and showing has differential protein to express band, the about 15KDa of molecular weight, consistent with theoretical value.Expression product has the activity that short 1/3 liver divides excision rat hepatocytes propagation equally

6. anti-liver injury activity

Through trial test, with mortality ratio, pathological change and liver function are the selected 4ml/kgCCL4 of index comprehensive (1: 1 CCL4: soya-bean oil) be best lethal dose.Get 60 of male Wister big white mouse, body weight 200 ± 15g, press 4ml/kg disposable celiac injection CCL4 for every, after 4 hours, be divided into 3 groups at random, every group of 20 rats, disposable celiac injection positive colony transforms cos-7 cell pyrolysis liquid 1ml respectively, and physiological saline 1ml and lotus empty plasmid transform cos-7 cell conditioned medium and lysate 1ml, write down dead animal day by day, survival surpasses 120h and counts surviving animals, two groups of control group survival rates of result are 15%, and positive colony group survival rate reaches 40%, in addition, we also observe the reorganization liver proliferating agent and have the effect that reduces poisoning rat peripheral blood transaminase level, show that the reorganization liver proliferating agent has the activity of anti-liver injury.

In a word, the invention discloses a kind of nucleotide sequence of human liver proliferating agent, and the physico-chemical property (is 15KDa as heat-resisting, molecular weight) and the biological activity assay method (as: biological activity in the body) of expression product are provided.This sequence can be reconstituted in any expression vector, to carry out the expression of protokaryon, eucaryon (as bacterium, yeast, insect, high equivalent thing, the mammalian cell of cultivation etc.).

CDNA of the present invention and encoded polypeptides product thereof can by with detectable (as ³²P-dATP, ¹²⁵I etc.) mark, and become a kind of reagent, being used for fixing tissue, the detection of liquid sample and DNA hybridization are to determine the location of genes involved in the human liver proliferating agent assignment of genes gene mapping and the map.

Because the recombinant human liver proliferating agent has the activity that promotes hepatocyte growth and anti-liver injury, therefore recombinant polypeptide product of the present invention or be furnished with thinner, the goods of assistant agent etc. may be treated serious hepatopathy (as Severe Viral Hepatitis) by applying clinical.

Claims

1. nucleotide sequence of human liver proliferating agent polypeptide of encoding, it has accompanying drawing 1 listed dna sequence dna.

2. according to the described nucleotide sequence of claim 1, it comprises the complementary strand of listed dna sequence dna in the accompanying drawing 1, and the gene order and the dna artificial sequence synthetic of can encode human liver proliferating agent and its analogue.

3. human liver proliferating agent, it can promote hepatocyte growth and have to improve the effect that CCL4 induces the acute liver failure rats survival rate, and its molecular weight is 15KDa, and contains aminoacid sequence shown in the drawings attached 2.

4. the method for a recombinant production human liver proliferating agent is characterized in that the described dna sequence dna of claim 1-2 is placed suitable carrier, transforms protokaryon or eukaryotic host cell, cultivates transformant under conditions suitable, obtains the human liver proliferating agent polypeptide

5. according to the described method of claim 4, wherein suitable carrier is a prokaryotic expression carrier, and wherein prokaryotic hosts is intestinal bacteria.

6. according to the described method of claim 4, wherein eukaryotic host cell is a mammalian cell.