CN1490410A - High effective expression of recombined human internal inhibition factor - Google Patents

Abstract

An efficient expression method of recombinant human internal inhibin features that the recombinant human internal inhibitin is induced by lactose to efficiently express it in the engineered colibacillus strain containing the nucleotide sequence shown in SEQ ID No.3. The application of said nucleotide sequence in configuring the engineered colibacillus strain able to efficiently express the recombinant human internal inhibin and the application of lactose induction method in inducing the efficient expression of recombinant human internal inhibitin are also disclosed.

Description

The high-efficiency expression method of chalone in the recombinant human

Technical field

The present invention relates to the genetically engineered field.More specifically, the present invention relates to utilize lactose to induce chalone in the recombinant human to efficiently express the method for chalone in the production recombinant human in the colibacillus engineering strain as inductor, wherein this project bacterial strain contains just like SEQ ID No 3 and nucleotide sequence shown in Figure 3; And chalone and be used for anti-neovascularization in the said gene engineering method recombinant human of being produced, the particularly purposes in the anti-tumor drug in preparation; In addition, the invention still further relates to SEQ ID No 3 and nucleotides sequence shown in Figure 3 and be listed in purposes in the colibacillus engineering strain that makes up chalone in the efficiently expressing recombinant human; And lactose-induced method purposes in the efficiently expressing of chalone in inducing recombinant human.

Background technology

Interior chalone is 183 amino acid polypeptide products of C-terminal behind interior collagen 18 natural degradations of organism, can be as detecting in blood, the urine in biological fluid.Molecular weight 20-24kDa size.O ' Reilly in 1997 ^[1]Find this a part Deng report at first, and point out this a part,, also have the effect that obvious inhibition mouse transplanting tumor is grown so have anti-new vessel formation function because of having the effect that suppresses vascular endothelial cell growth.At Boehm ^[2]Deng research in find that the interior chalone molecule after the application of purified not only obviously suppresses the growth of mouse transplanting tumor repeatedly, and have no side effect, do not produce the resistance of mouse yet, this is one of most promising developing drugs of a treatment tumour beyond doubt.Only after 2 years, before the clinical experiment of U.S. FDA with chalone treatment noumenal tumour in the fastest in history speed approval recombinant human, chalone is in II phase clinical experiment in the recombinant human.

At present, in the clinical experiment in the employed recombinant human chalone be solubility, produce by yeast expression because cost and output are not high, people also seek new efficiently express, chalone in low-cost, the simple approach production of the process recombinant human.1997, in O ' Reilly finds behind the chalone, very fast chalone gene clone in the mouse is gone in the prokaryotic system expression vector, and chalone efficiently expresses in the acquisition recombined small-mouse, its induction method is to utilize IPTG, and the albumen after the expression exists with the inclusion body form, but albumen is difficult to exist with soluble form, utilize renaturation folding process processing again, soluble proteins less than 1%.Be difficult in expression in escherichia coli goes out soluble recombined human behind the chalone, people inquire into various ways, the expression of chalone in the recombinant human of number of different types, and the interior chalone that has obtains high expression level but is insoluble or do not possess activity, possessing of having is active insoluble, have solvable but output is not high.As Boehem ^{[3] [4]}Deng chalone in the successful expression solubility mouse in Yeast system in 1998, but obtained product has inhomogenous N end, analysis may be the reason owing to proteasome degradation, chalone in the mouse of B16F10 melanoma cell express recombinant, obtain homogeneous N end product, activity is arranged, but output is very low.SaSaki in 1998 ^[5]Utilize the interior chalone of HEKC express recombinant people can obviously suppress the growth of mouse transplanting tumor.In this system, expressed protein yield is also very limited.Dhanabal in 1999 ^[6]Deng utilizing escherichia coli high-level expression to go out chalone in the recombined small-mouse, but albumen is insoluble, and the expressed albumen that goes out has monomer, and molecular weight 22-25kDa also has the binary molecule, and molecular weight is at 44-46kDa.From employed expression system analysis, the expression amount that still is bacterium is for the highest, but solvability is an a great problem, and the soluble approach of chalone in the reorganization that can make bacterial expression is being sought always by many study group ^[7], point out that from institute's acquisition data as inducing, domestic also have employing temperature control and osmotic pressure change to induce the report that chalone is expressed in the recombinant human to the chalone major part with IPTG in the bacterial expression reorganization ^[8]

Therefore, ability presses for a kind of can efficiently expressing, and can make in the expressed reorganization chalone solvable and have in the people method of chalone in the active production recombinant human of chalone institute inherent again.

Summary of the invention

At above-mentioned needs of the prior art, the inventor is through long-term exploration and a large amount of research, finally successfully invented a kind of can efficiently expressing, can make in the expressed reorganization chalone solvable and have in the people method of chalone in the active production recombinant human of chalone institute inherent again, thereby solve long-term puzzlement those skilled in the art's technical barrier: the present invention is according to the Nucleotide of the terminal encoding amino acid sequence of people's collagen 18C ^[9]The PCR primer of chalone in the synthetic amplification recombinant human, utilize people's embryonic liver cell mRNA as template then, through reverse transcription again behind the pcr amplification (RT-PCR), obtain desired amplified fragments, through nucleotide sequencing, in the gene that obtains and the people who is inferred the chalone gene order be consistent, for making gene in intestinal bacteria, obtain to efficiently express, under the prerequisite that does not change aminoacid sequence, design the N end, 4 amino acid coding of bacterium preference, after expression vector pET 24b (the .cat No.69418-3 of Novagen company) is gone in the chalone gene clone in the recombinant human after the sudden change, can induce the expression that produces chalone in the recombinant human through IPTG, because the expression process has leakage phenomenon, the present invention utilizes the leakage of glucose control protein expression, utilize lactose-induced proteic expression then, the expression of chalone in bacterium accounts for about 40% of tropina in the recombinant human as a result, fermenting experiment is amplified in pilot scale, lactose can successfully be induced the expression of chalone in the recombinant human, about expression amount 40%, and be easy to dissolving through the recombinant human endostatin protein of lactose-induced expression, purifying and renaturation (solvability is fine), the albumen that obtains have the effect that suppresses endothelial cell growth and animal-transplanted tumor growth significantly.One of purpose of the present invention is to utilize lactose as inductor, induces chalone efficiently expressing in intestinal bacteria in the recombinant human; Utilize lactose as inductor, in pilot scale and scale operation, induce efficiently expressing of chalone in the recombinant human.

Therefore, first aspect of the present invention provides a kind of method for preparing chalone in the recombinant human, it is characterized in that using the high expression level bacterium engineering strain that contains the nucleic acid shown in the SEQ ID No.3 (accompanying drawing 3) to carry out.Preferably, in the method, utilize lactose, induce chalone efficiently expressing in intestinal bacteria in the recombinant human as inductor.More preferably, in the method, utilize the leakage (leakeage refers to non-inducible expression) of glucose control protein expression.

The expression vector that second aspect of the present invention, a kind of big enterobacteria engineering strain that provides, this bacterial strain have been contained the nucleic acid shown in the SEQ ID No.3 (perhaps accompanying drawing 3) transforms.Chalone in this coli strain energy efficiently expressing recombinant human, and the aminoacid sequence of this recombinant protein is shown in SEQ ID No.4 (perhaps accompanying drawing 3).Preferably, this big enterobacteria engineering strain is to be preserved in Chinese typical culture collection center (CCTCC, Wuhan) and preserving number is intestinal bacteria BL-21 (DE-3)/pENDO engineering strain of CCTCC NO:M203063 on August 6th, 2003.

The 3rd aspect of the present invention, chalone in a kind of recombinant human is provided, it is to be produced by aforesaid method, its sequence is shown in SEQ ID No.4 (perhaps accompanying drawing 3), (solvability is fine to be easy to purifying and renaturation, Yi Rong) and with the people in chalone have identical biological function, promptly have the function that suppresses vascular endothelial cell growth, thereby treatment and prophylactic effect arranged because of the disease due to the neovascularization various.

The 4th aspect of the present invention provides SEQ ID No 3 and nucleotide sequence shown in Figure 3.

The 5th aspect of the present invention provides SEQ ID No 3 and nucleotides sequence shown in Figure 3 to be listed in purposes in the colibacillus engineering strain that makes up chalone in the efficiently expressing recombinant human.

The 6th aspect of the present invention provides lactose-induced method purposes in the efficiently expressing of chalone in inducing recombinant human.

The 7th aspect of the present invention, the interior chalone of the recombinant human that provides the said gene engineering method to be produced is used for anti-neovascularization in preparation, particularly the purposes in the anti-tumor drug.

But in following " detailed Description Of The Invention ", especially on the basis of the disclosure of " embodiment " part, other aspects and advantages of the present invention are conspicuous to those skilled in the art.

Description of drawings:

Fig. 1: people's collagen 18 Nucleotide and aminoacid sequence (nucleic acid and the aminoacid sequence of chalone (Endostatin) in the people).

Fig. 2: behind the pcr amplification chalone gene order figure in the people that surveys, Fig. 2-1:5 ' direction wherein, Fig. 2-2:3 ' direction.

Fig. 3: chalone nucleotide sequence and aminoacid sequence in the sudden change descendant.

Fig. 4: the interior chalone gene order figure of the people that surveys of sudden change back institute.

Fig. 5: chalone gene agarose gel electrophoresis figure in the pcr amplification people: swimming lane 1: standard nucleotides molecular weight; 2,4: do not contain magnesium ion in the amplified reaction; 3,5: contain the 1mM magnesium ion in the amplified reaction; 6:50 ℃ of amplification contains the 4mM magnesium ion; 7:58 ℃ of amplification contains the 4mM magnesium ion.

Fig. 6: pET 24b expression vector figure (recombinant human vascular endothelial inhibin plasmid construction collection of illustrative plates) is gone in the chalone gene clone in the recombinant human.

Fig. 7: chalone IPTG abduction delivering in intestinal bacteria BL-21 (DE3) in the recombinant human: swimming lane 1: do not induce (containing glucose suppresses); 2: do not induce (not containing glucose suppresses); 3:IPTG induced 2 hours; 4:IPTG induced 3 hours; 5:IPTG induced 4 hours.

Fig. 8: chalone lactose-induced expression in intestinal bacteria BL-21 (DE3) in the recombinant human: swimming lane 1: do not induce; 2-8: be respectively and induce 2,4,6,8,10,12,14 hours, the scanning result of expression amount is 8, expression amount: 42.0171/99.7967 * 100%=42.1%.

Fig. 9: lactose-induced expression in the fermentation is amplified in the chalone pilot scale in the recombinant human: swimming lane 1: blank; 2: do not induce; 3-9: be respectively and induce 2,4,6,8,10,12,14 hours; 10: standard molecular weight.The expression amount scanning result is 9 roads, expression amount: 39.5012/99.8929 * 100%=39.5%.

Figure 10: chalone SDS-gel electrophoresis figure in the recombinant human behind the purification renaturation: swimming lane 1: reference substance; The 2:CM purification of samples; The 3:CM purification of samples.The purity scanning result is 3 roads, and purity is 99.0124/99.7281 * 100%=99.3%.

Figure 11: the restraining effect of after the chalone renaturation human endothelial cell being grown in the recombinant human: the longitudinal axis is inhibiting rate (%), and transverse axis is interior chalone concentration.IC ₅₀≤5μg。

Figure 12: chalone is to the restraining effect of the growth of mice-transplanted tumor in the recombinant human: the longitudinal axis is transplanted tumor volume (mm ³), the transverse axis time (my god).

Detailed Description Of The Invention

In the context of the present specification, unless specialize, otherwise the used any technical term of this specification sheets has those of ordinary skills' implication of common sense in the art, and the experimental technique of unreceipted actual conditions is according to the normal experiment method, translate molecular cloning experiment guide second edition, Science Press as Jin Dongyan etc., Beijing, 1992; Wu Naihu etc., Principles of Gene Engineering, Science Press, Beijing, 2000; The thick plinth of Zhu etc. is translated protein purification and identification experiment guide, Science Press, Beijing, 1999; With Li Yuyang chief editor, gene expression technique, Science Press, Beijing, 2000 described carrying out; Or carry out according to the process specifications that supplier advised.

For obtaining chalone gene in the people, according to terminal 183 aminoacid sequences (Fig. 1) of people's collagen 18C, synthetic N end and C end primer sequence, and at N end design of primers NdeI restriction enzyme site and initial code ATG, C end design of primers adds after stop code-the BamHI site, personnel selection embryonic liver cell mRNA is as template, chalone gene in the RT-PCR method amplification people, through the agarose gel electrophoresis analysis, the about 550bp molecular weight of amplified fragments size, with be consistent (Fig. 5) of estimating, after gene fragment clone gone into Topo PCR2.1 cloned plasmids (invitrogen company), amplification and plasmid purification, through nucleotide sequence analysis, institute's gene order that obtains and people's collagen 18C end 183 aminoacid sequences (Fig. 2) in full accord, again gene clone is gone among the prokaryotic system expression vector plasmid pET 24b (available from Novagen company), be transformed in BL-21 (DE3) the host bacterium (available from Invitrogen company), after the cultivation, induce the expression of chalone in the recombinant human with IPTG, express the back and collect bacterium, behind the SDS-polyacrylamide gel electrophoresis, one obvious abduction delivering band is arranged about molecular weight 20kDa size, expression amount about about 20%, for improving the expression of chalone in prokaryotic cell prokaryocyte in the recombinant human, the present invention designs 1 at the interior chalone gene N end of mutant human, the nucleotide coding of 3-5 amino acids, make it to become bacterial ribosome identification preference (Fig. 3, Fig. 4), to in the recombinant human after the sudden change, be cloned into again in the pET 24b plasmid by the chalone gene, be transformed among the BL-21 (DE3), induce through IPTG, the recombinant human endostatin protein is expressed and is brought up to about 40% (Fig. 7), find in the experiment in the control group without lactose-induced expression, chalone expression in the people about 10% is also arranged, illustrate that the expression of this plasmid has leakage phenomenon.Before the present invention adopts not abduction delivering, can successfully stop this leakage with adding glucose in the substratum, and utilize lactose again can its expression amount of successful abduction delivering about 40%, in fermentor tank pilot scale amplification test, the present invention adds glucose in prior fermentation, both can be used as carbon source, and can effectively control again to express and reveal, treated that bacterium generated density and reaches OD ₆₀₀During the 14-18 left and right sides, add lactose and induce, continue culturing bacterium about 16 hours, bacterial density can reach OD ₆₀₀More than 20, the chalone expression amount is about 40% (Fig. 9) in the recombinant human, after lactose-induced, chalone extracts through inclusion body in the recombinant human, steps such as renaturation purifying, can obtain purity and reach more than 95%, and the soluble proteins more than 99% (Figure 10) preferably, the albumen behind the purifying has the effect (Figure 11 and 12) of obvious suppression human vascular endothelial and mice-transplanted tumor growth.

Below in conjunction with specific embodiment, further illustrate the present invention.Should be understood that these embodiment just in order to demonstrate the invention, but not limit the scope of the invention by any way.In the following example, the experimental technique of unreceipted actual conditions carries out according to the normal experiment method usually, or carries out according to the operation instructions that manufacturer advises.Embodiment 1: the acquisition of chalone gene and the sudden change of bacterium preference type gene in the recombinant human

According to 183 amino acid whose nucleotide sequences of people's collagen 18C end, the primer of at first synthetic RT-PCR, its primer sequence is as follows:

5’-GC?CAT?ATG?CAC?AGC?CAC?CGC?GAC-3’

3 ' end primer 5 '-CG GGA TCC CTA CTT GGA GGC AGT-3 '

People's embryonic liver cell mRNA derives from people's embryo hepatic tissue, is extracted by Trizol reagent (Invitrogen company) single stage method.

RT-PCR reaction conditions following (the RT-PCR test kit is available from Roche company):

1. 5 * reverse transcription damping fluid, 4 μ l

MgCl ₂ 2μl(50mM)

dNTP 2μl(10mM)

Random primer 1 μ l (50pmol)

ThermoScript II 1 μ l 2.5IU

mRNA 2μl(1μg/μl)

RNase inhibitor 1 μ l

42 ℃ of water-baths are after 100 minutes, and 95 ℃ are incubated 5 minutes ,-80 ℃ of preservations.

2. template (cDNA reaction solution) 5 μ l

5 ' primer 2 μ l 50pmole

3 ' primer 2 μ l 50pmole

dNTP 2μl 10mM

5 * PCR damping fluid, 20 μ l

MgCl ₂ 2μl 100mM

Taq archaeal dna polymerase 1 μ l 5U

H ₂O 76μl

End reaction volume 100 μ l

The condition of polymerase chain amplified reaction (PCR) is as follows: 98 ℃ of sex change 5 minutes, and 94 ℃ 40 seconds, 56 ℃ 40 seconds, 72 ℃ totally 30 circulations in 50 seconds then, last 72 ℃ prolong 10 minutes.After reaction finishes, the PCR sample placed on ice or-80 ℃ frozen.

The reacted sample of RT-PCR in 1% agarose electrophoresis, as seen there is obvious amplification gene fragment (Fig. 5) at 550bp molecular weight size place.Get 1 μ l amplification reaction solution, mix 3 μ lH ₂O and 1 μ l Topo PCR 2.1 carriers (Invitrogen company) mixed solution, effect is after 5 minutes under the room temperature, get 1 μ l and put into competence bacterium shot one (Invitrogen company) 50 μ l, behind the ice bath 30 minutes, 42 ℃ of water-baths 30 seconds, add＞50 μ l SOS (Invitrogen company) substratum (purchasing company) again in Invitrogen, 37 ℃ of water-baths 30 minutes, the LB agar plate that kantlex (30 μ g/ml) is contained in the shop spends the night, 37 ℃, selected mono-clonal in second day, after LB (containing kantlex 30 μ g/ml) substratum jolting is cultivated, extract plasmid, enzyme is cut evaluation.Do nucleotide sequence analysis simultaneously, the result confirms, the gene order that obtains and the sequence (Fig. 2) in full accord inferred, utilize N end NdeI and C end BamHI restriction enzyme site, downcut the interior chalone gene of recombinant human and be cloned into (Fig. 6) among the expression vector pET24b, behind transformed into escherichia coli cell BL-21 (DE3), in the LB that contains 50 μ g/ml penbritins, cultivate and induce the expression of chalone in the recombinant human, as bacterial concentration OD ₆₀₀When reaching 0.6-0.8, add IPTG to final concentration be after 0.5mM induces 4 hours, centrifugal collection bacterium, through after the bacteria breaking under the 15%SDS-polyacrylamide gel electrophoresis, detect the expression of chalone in the recombinant human, the result is presented at about molecular weight 20kDa, one obvious protein induced expression band is arranged, scanning method calculation expression amount is about 20%, for further improving the expression of chalone in intestinal bacteria in the recombinant human again, utilize the method for PCR, the encoding mutant of preceding 4 human amino acids of chalone gene in the people is become bacterium preference type.It is as follows that 5 ' distal process becomes primer: 5 '-GC CAT ATGCAT AGC CAT CGT GAT TTC CAG CCG GTG CTCC-3 ' and former 3 ' end combination of primers, utilize chalone gene plasmid in the wild-type people (above-mentioned 550bp PCR fragment pack into Topo PCR2.1 carrier) to be template, after same PCR method (except that the primer difference), also obtain the 550bp amplified fragments, be cloned into Topo PCR 2.1(Invitrogen) after, extract plasmid and do nucleotide sequence analysis, its result with estimate sudden change result be consistent (Fig. 4), chalone gene in the recombinant human after the sudden change is cloned among the pET 246 again, after transforming BL-21 (DE3), after cultivating the IPTG abduction delivering, after collecting thalline 15%SDS-polyacrylamide gel electrophoresis, 40% (Fig. 7) that is about bacterioprotein at the expression band of 20kDa molecular weight size, the result confirms that mutant plasmid is 2 times of the expression of wild type gene cloned plasmids in bacterium.Preferably, this big enterobacteria engineering strain is to be preserved in Chinese typical culture collection center (CCTCC, Wuhan) and preserving number is intestinal bacteria BL-21 (DE-3)/pENDO engineering strain of CCTCC NO:M203063 on August 6th, 2003.

Embodiment 2: chalone efficiently expressing in intestinal bacteria in the lactose-induced recombinant human

The mutant plasmid is at BL-21 (DE ₃But) the interior chalone of host bacterium inducement efficient express recombinant people.And in experiment, find, even tangible protein expression is also arranged without IPTG inductive control group.Illustrate because promotor is strong excessively, expression to chalone in the people has the startup leakage phenomenon, the present invention once utilized different temperature, different time, different substratum to attempt preventing to reveal, find to utilize glucose can effectively prevent to reveal at last, and not only induce and express chalone in the recombinant human down at IPTG, and utilize lactose equally can the effective expression recombinant human in chalone, expression amount reaches about 40% (Fig. 8).

It is as follows that concrete substratum is formed part:

Select mono-clonal, contain among the fresh LB of 50 μ g penbritin 1ml at 1.5ml, 37 ℃ of overnight incubation, second day, will spend the night bacterium with 1: 50 fresh LB (containing the ammonia benzyl) dilution, 37 ℃ of joltings are cultured to bacterium OD again ₆₀₀1.2 about, adding lactose to final concentration is 2%, continues culturing bacterium 6-8 after individual hour, the expression of chalone in the centrifugal collection Bacteria Detection recombinant human.

Embodiment 3: chalone efficiently expressing in pilot scale fermentation in the lactose-induced recombinant human

Chalone fermentation using bacteria seed liquor is composed as follows in the recombinant human: contain the 10g casein in every liter of substratum, 5g yeast extract powder, 10g sodium-chlor, 2% glucose.Actual mechanical process is as follows: the fresh single bacterium colony of picking, be inoculated in the 200ml fresh seeds liquid, and incubated overnight, 10 times of volume fresh seeds liquid continued enlarged culturing to bacterium OD in second day ₆₀₀2.0 during the left and right sides, add in the fermentor tank, ratio is amplification in 1: 10, the substratum in the fermentor tank still is the seed liquor composition.In the fermenting process, when dissolved oxygen reduces to 0, add the feed supplement 1 of 1/10 volume, composition is a glucose, and sulfuric acid amine and sal epsom are respectively 20%, 10% and 1%, as the bacterium OD that cultivates ₆₀₀Reach at 10 o'clock, add 10% yeast extract of 1/20 volume, work as OD ₆₀₀When rising to 14 left and right sides, add ultimate density and be 2% lactose liquid and carry out abduction delivering, continue to cultivate after about 14 hours OD again ₆₀₀During about 20 left and right sides, put jar, the wet bacterium 40g of every liter of fermented liquid Yue Kede this moment, bacterial expression amount about 40%, Fig. 9 is the expression of chalone in the recombinant human under the fermentation different time.

Example 4. adopts and example 3 identical methods, only changes lactose concn, is respectively 0.01%, 0.5%, 1%, 10%, measures the bacterial expression amount, is respectively 10%, 20%, 40%, 36%.

Embodiment 5: chalone is through inclusion body purification, renaturation, repurity in the lactose-induced recombinant human

After collecting thalline, every jar of fermented liquid can obtain wet bacterium 1,400g, buffer A (50mM Tris-HCl, pH8.3,1mM EDTA with ice bath, the 1mM beta-mercaptoethanol), add final concentration behind about 7500ml suspendible thalline again and be 0.05% N,O-Diacetylmuramidase, 4 ℃ of following carrying out ultrasonic bacteria breaking, ultrasonic power is 1,000W, time 10min, suspended 3 seconds in ultrasonic 10 seconds, behind the ultrasonic degradation, the smear test under microscope, the bacteria breaking rate answers＞95%.4 ℃ of following bacterial lysates are centrifugal 7,000rpm/min, and centrifugal 20 minutes, abandon supernatant, precipitation is thick inclusion body.With buffer B (50mM Tris-HCl, pH8.3,4M urea, 1%Trition X-100,1mM EDTA, 5mM beta-mercaptoethanol), behind the suspendible inclusion body, put room temperature following 10 minutes, centrifugal 7, the 000rpm/ branch, 30 minutes, wash 1 time, with the buffer A washing once, the purity of inclusion body can bring up to 75% from 50% again.With damping fluid C (50mM Tris-HCl, pH8.3,8M urea, 1mM EDTA, 15mM beta-mercaptoethanol), 4 ℃ of following stirring and dissolving inclusion bodys, every gram weight in wet base inclusion body dissolves with 20ml damping fluid C.After stirring and dissolving is spent the night, 10, centrifugal 30 minutes of 000rpm/min collects supernatant, abandons precipitation (supernatant is solubilization of inclusion bodies liquid).

Will (molecular retention be 12 in the interior chalone behind the DEAE-Sepharose Fast Flow purifying is packed dialysis tubing into, 000-14,000kDa), under the PBS damping fluid, add 4M urea, dialysed 24 hours, subtracting urea again is 2M, regulate pH to 7.0, add 0.1mM Sleep-promoting factor B and 1mM reduced glutathion simultaneously, dialysed 24 hours, with balance liquid (20mMNaAc/Ac, pH5.8) balance columns bed is gone up sample then, uses the online detection of nucleic acid-protein detector (detecting wavelength 280nm) simultaneously, use elutriant A (0.1M NaCl behind the last sample, 20mM NaAc/AcpH5.8) wash-out is collected elution peak A, cleans with balance liquid again, use elutriant B (0.3MNaCl at last, 20mM NaAc/Ac, pH5.8) wash-out is collected elution peak B, identify that through the SDS-PAGE electrophoresis elution peak B contains endostatin protein, the pure product purity that obtains after the purified renaturation is 99.3%.As shown in figure 10.

Embodiment 6: the restraining effect of after the chalone renaturation human endothelial cell being grown in the recombinant human

Select the vigorous ECV cell (ATCC) of growth conditions, add a small amount of pancreatin, in room temperature digestion, treat digestion fully after, carefully remove pancreatin, add and contain 3 μ g.L ^-1The RPMI medium 1640 of bFGF and 2%FBS and penicillin 10 ⁵U.L ^-1, Streptomycin sulphate 100mg.L ^-1The substratum termination reaction, with dropper cell on the bottle wall is blown down from wall, and piping and druming is evenly.Adjusting cell concn again is every milliliter 2 * 10 ⁴Individual, the cell suspension of adding 0.1ml in 96 orifice plates.Chalone carries out different concns gradient doubling dilution with above-mentioned substratum again in the recombinant human, and each concentration gradient is provided with 3 multiple holes, and every hole liquid feeding 0.1ml makes final concentration (mg.L ^-1) be respectively: 100,50,25,12.50,6.25,3.12,1.56,0.78, blank group and negative control group are set simultaneously.At 37 ℃, saturated humidity and 5%CO ²Static cultivation 4h under the condition.Behind the 4h, the careful suction gone culture supernatant, adds 0.15ml DMSO, and dissolved cell shakes up with the culture plate electromagnetic shaker.Be 630nm with microplate reader by reference wavelength again, the test wavelength is that 492nm measures each hole OD absorption value, obtain the mean value that each extent of dilution 3 hole of blank and sample record, each extent of dilution average light of sample absorbed deducting blank average light and absorb, is 50% o'clock sample concentration with the absorbance of negative control.

The inhibiting rate of chalone calculates by following formula in the recombinant human:

According to the inhibiting rate of each point, draw amount effect curve: with chalone dosage in the recombinant human is X-coordinate, is ordinate zou with the inhibiting rate, Microsoft Excel mapping, and observing the restraining effect of chalone in the recombinant human and asking inhibiting rate is 50% o'clock formulation concentrations.

The result shows: the concentration that produces the interior chalone of recombinant human of 50% endotheliocyte inhibition is about 3.50mg.L ^-1See Figure 11.

Embodiment 7: chalone is to the restraining effect of the growth of mice-transplanted tumor in the recombinant human

Test method:

1. animal Kunming mouse (China Medicine University experimental animal center), male and female dual-purpose, body weight 18-22g, mouse 5-6 in age week.

2. tumor model S ₁₈₀Sarcoma.

3. tumour transplatation selects tumor growth vigorous and do not have the tumor-bearing mice of diabrosis, and the cervical vertebra dislocation is put to death, and under aseptic condition (super clean bench), with the tincture of iodine, alcohol disinfecting animal skin, cuts skin, peels off tumour and weighs.In culture dish, tumor tissue is cut into small pieces (room temperature＞15 ℃ time should put on the ice cube operate), puts into aseptic homogenizer, add sterile saline, homogenate by 1: 3～1: 4 (W/V).Get and be subjected to the knurl mouse, in the forelimb armpit place tincture of iodine, alcohol disinfecting, subcutaneous injection tumour homogenate 0.2ml/ is (tumor cell number about 5 * 10 only ⁶Individual).

4. animal divides into groups in inoculating back the 3rd day tumor-bearing mice to be divided into negative control group, positive controls and treatment group at random.General 10 of treatment treated animal number, negative control level number of animals is treatment treated animal * preparation of √ medicine and test group number.

5. medicine preparation and the water-soluble solid pharmaceutical of route of administration are prepared with physiological saline or water for injection.Subcutaneous injection administration (must note the sterilization of injection site), be administered once every day, negative control group injection equal-volume physiological saline or water for injection.

6. touch the part every day behind the administration time inoculated tumour, begins administration when diameter of tumor reaches 1～2mm, continuous use 7 days.

7. measure the major diameter (r of tumour after the observation index administration every day ₁), minor axis (r ₂), calculate tumor size (V=1/2 * r ₁* (r ₂) ²), and the record animal has no adverse reaction or dead, if there is death to write down the death time, analyzes the cause of death.

8. inhibition rate of tumor growth is according to above-mentioned measuring result, and (relative tumor volume, RTV), calculation formula is: RTV=V to calculate the tumour relative volume _t/ V ₀V wherein ₀Measure the gross tumor volume of gained during for administration, V _tGross tumor volume when measuring each time.The evaluation index of anti-tumor activity is relative tumor proliferation rate T/C (%), and calculation formula is as follows:

$T/C(\%)=\frac{T_{\mathrm{RTV}}}{C_{\mathrm{RTV}}}\×100\%$

Wherein: T _RTV: treatment group RTV; C _RTV: negative control group RTV.

The result shows: press 5mg/kg, chalone can obviously suppress the growth of people's cancer of the stomach BGC803 nude mice transplantation tumor in 10mg/kg and the 20mg/kg dosage subcutaneous injection recombinant human, as shown in figure 12.

Total the above, chalone is after inclusion body purification, renaturation, repurity in the lactose-induced recombinant human of the present invention, purity of protein (HPLC) reaches (Figure 10) more than 95%, and has tangible biological activity (Figure 11,12).

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Sequence table＜110〉Xinghu Biotech Co., Ltd., Zhaoqing City, Guangdong Prov.＜120〉high-efficiency expression method＜130〉I020301＜160〉4＜170〉PatentIn version 3.1＜210 of recombinant human endostatin〉1＜211〉552＜212〉DNA＜213〉Homo sapiens＜220〉＜221〉CDS＜222〉(1) .. (552)＜223〉＜400〉1cac agc cac cgc gac ttc cag ccg gtg ctc cac ctg gtt gcg ctc aac 48His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn1,5 10 15agc ccc ctg tca ggc ggc atg cgg ggc atc cgc ggg gcc gac ttc cag 96Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln

20 25 30tgc?ttc?cag?cag?gcg?cgg?gcc?gtg?ggg?ctg?gcg?ggc?acc?ttc?cgc?gcc 144Cys?Phe?Gln?Gln?Ala?Arg?Ala?Val?Gly?Leu?Ala?Gly?Thr?Phe?Arg?Ala

35 40 45ttc?ctg?tcc?tcg?cgc?ctg?cag?gac?ctg?tac?agc?atc?gtg?cgc?cgt?gcc 192Phe?Leu?Ser?Ser?Arg?Leu?Gln?Asp?Leu?Tyr?Ser?Ile?Val?Arg?Arg?Ala

50 55 60gac?cgc?gca?gcc?gtg?ccc?atc?gtc?aac?ctc?aag?gac?gag?ctg?ctg?ttt 240Asp?Arg?Ala?Ala?Val?Pro?Ile?Val?Asn?Leu?Lys?Asp?Glu?Leu?Leu?Phe65 70 75 80ccc?agc?tgg?gag?gct?ctg?ttc?tca?ggc?tct?gag?ggt?ccg?ctc?aag?ccc 288Pro?Ser?Trp?Glu?Ala?Leu?Phe?Ser?Gly?Ser?Glu?Gly?Pro?Leu?Lys?Pro

85 90 95ggg?gca?cgc?atc?ttc?tcc?ttt?gac?ggc?aag?gac?gtc?ctg?agg?cac?ccc 336Gly?Ala?Arg?Ile?Phe?Ser?Phe?Asp?Gly?Lys?Asp?Val?Leu?Arg?His?Pro

100 105 110acc?tgg?ccc?cag?aag?agc?gtg?tgg?cat?ggc?tcg?gac?ccc?aac?ggg?cgc 384Thr?Trp?Pro?Gln?Lys?Ser?Val?Trp?His?Gly?Ser?Asp?Pro?Asn?Gly?Arg

115 120 125agg?ctg?acc?gag?agc?tac?tgt?gag?acg?tgg?cgg?acg?gag?gct?ccc?tcg 432Arg?Leu?Thr?Glu?Ser?Tyr?Cys?Glu?Thr?Trp?Arg?Thr?Glu?Ala?Pro?Ser

130 135 140gcc?acg?ggc?cag?gcc?tcc?tcg?ctg?ctg?ggg?ggc?agg?ctc?ctg?ggg?cag 480Ala?Thr?Gly?Gln?Ala?Ser?Ser?Leu?Leu?Gly?Gly?Arg?Leu?Leu?Gly?Gln145 150 155 160agt?gcc?gcg?agc?tgc?cat?cac?gcc?tac?atc?ctg?ctc?tgc?att?gag?aac 528Ser?Ala?Ala?Ser?Cys?His?His?Ala?Tyr?Ile?Leu?Leu?Cys?Ile?Glu?Asn

165 170 175agc?ttc?atg?act?gcc?tcc?aag?tag 552Ser?Phe?Met?Thr?Ala?Ser?Lys

180<210>2<211>183<212>PRT<213>Homo?sapiens<400>2His?Ser?His?Arg?Asp?Phe?Gln?Pro?Val?Leu?His?Leu?Val?Ala?Leu?Asn1 5 10 15Ser?Pro?Leu?Ser?Gly?Gly?Met?Arg?Gly?Ile?Arg?Gly?Ala?Asp?Phe?Gln

20 25 30Cys?Phe?Gln?Gln?Ala?Arg?Ala?Val?Gly?Leu?Ala?Gly?Thr?Phe?Arg?Ala

35 40 45Phe?Leu?Ser?Ser?Arg?Leu?Gln?Asp?Leu?Tyr?Ser?Ile?Val?Arg?Arg?Ala

50 55 60Asp?Arg?Ala?Ala?Val?Pro?Ile?Val?Asn?Leu?Lys?Asp?Glu?Leu?Leu?Phe65 70 75 80Pro?Ser?Trp?Glu?Ala?Leu?Phe?Ser?Gly?Ser?Glu?Gly?Pro?Leu?Lys?Pro

85 90 95Gly?Ala?Arg?Ile?Phe?Ser?Phe?Asp?Gly?Lys?Asp?Val?Leu?Arg?His?Pro

100 105 110Thr?Trp?Pro?Gln?Lys?Ser?Val?Trp?His?Gly?Ser?Asp?Pro?Asn?Gly?Arg

115 120 125Arg?Leu?Thr?Glu?Ser?Tyr?Cys?Glu?Thr?Trp?Arg?Thr?Glu?Ala?Pro?Ser

130 135 140Ala?Thr?Gly?Gln?Ala?Ser?Ser?Leu?Leu?Gly?Gly?Arg?Leu?Leu?Gly?Gln145 150 155 160Ser?Ala?Ala?Ser?Cys?His?His?Ala?Tyr?Ile?Leu?Leu?Cys?Ile?Glu?Asn

165 170 175Ser?Phe?Met?Thr?Ala?Ser?Lys

180<210>3<211>552<212>DNA<213>Artificial<220><221>CDS<222>(1)..(552)<223><400>3cat?agc?cat?cgt?gat?ttc?cag?ccg?gtg?ctc?cac?ctg?gtt?gcg?ctc?aac 48His?Ser?His?Arg?Asp?Phe?Gln?Pro?Val?Leu?His?Leu?Val?Ala?Leu?Asn1 5 10 15agc?ccc?ctg?tca?ggc?ggc?atg?cgg?ggc?atc?cgc?ggg?gcc?gac?ttc?cag 96Ser?Pro?Leu?Ser?Gly?Gly?Met?Arg?Gly?Ile?Arg?Gly?Ala?Asp?Phe?Gln

20 25 30tgc?ttc?cag?cag?gcg?cgg?gcc?gtg?ggg?ctg?gcg?ggc?acc?ttc?cgc?gcc 144Cys?Phe?Gln?Gln?Ala?Arg?Ala?Val?Gly?Leu?Ala?Gly?Thr?Phe?Arg?Ala

35 40 45ttc?ctg?tcc?tcg?cgc?ctg?cag?gac?ctg?tac?agc?atc?gtg?cgc?cgt?gcc 192Phe?Leu?Ser?Ser?Arg?Leu?Gln?Asp?Leu?Tyr?Ser?Ile?Val?Arg?Arg?Ala

50 55 60gac?cgc?gca?gcc?gtg?ccc?atc?gtc?aac?ctc?aag?gac?gag?ctg?ctg?ttt 240Asp?Arg?Ala?Ala?Val?Pro?Ile?Val?Asn?Leu?Lys?Asp?Glu?Leu?Leu?Phe65 70 75 80ccc?agc?tgg?gag?gct?ctg?ttc?tca?ggc?tct?gag?ggt?ccg?ctc?aag?ccc 288Pro?Ser?Trp?Glu?Ala?Leu?Phe?Ser?Gly?Ser?Glu?Gly?Pro?Leu?Lys?Pro

85 90 95ggg?gca?cgc?atc?ttc?tcc?ttt?gac?ggc?aag?gac?gtc?ctg?agg?cac?ccc 336Gly?Ala?Arg?Ile?Phe?Ser?Phe?Asp?Gly?Lys?Asp?Val?Leu?Arg?His?Pro

100 105 110acc?tgg?ccc?cag?aag?agc?gtg?tgg?cat?ggc?tcg?gac?ccc?aac?ggg?cgc 384Thr?Trp?Pro?Gln?Lys?Ser?Val?Trp?His?Gly?Ser?Asp?Pro?Asn?Gly?Arg

115 120 125agg?ctg?acc?gag?agc?tac?tgt?gag?acg?tgg?cgg?acg?gag?gct?ccc?tcg 432Arg?Leu?Thr?Glu?Ser?Tyr?Cys?Glu?Thr?Trp?Arg?Thr?Glu?Ala?Pro?Ser

130 135 140gcc?acg?ggc?cag?gcc?tcc?tcg?ctg?ctg?ggg?ggc?agg?ctc?ctg?ggg?cag 480Ala?Thr?Gly?Gln?Ala?Ser?Ser?Leu?Leu?Gly?Gly?Arg?Leu?Leu?Gly?Gln145 150 155 160agt?gcc?gcg?agc?tgc?cat?cac?gcc?tac?atc?ctg?ctc?tgc?att?gag?aac 528Ser?Ala?Ala?Ser?Cys?His?His?Ala?Tyr?Ile?Leu?Leu?Cys?Ile?Glu?Asn

165 170 175agc?ttc?atg?act?gcc?tcc?aag?tag 552Ser?Phe?Met?Thr?Ala?Ser?Lys

180<210>4<211>183<212>PRT<213>Artificial<400>4His?Ser?His?Arg?Asp?Phe?Gln?Pro?Val?Leu?His?Leu?Val?Ala?Leu?Asn1 5 10 15Ser?Pro?Leu?Ser?Gly?Gly?Met?Arg?Gly?Ile?Arg?Gly?Ala?Asp?Phe?Gln

20 25 30Cys?Phe?Gln?Gln?Ala?Arg?Ala?Val?Gly?Leu?Ala?Gly?Thr?Phe?Arg?Ala

35 40 45Phe?Leu?Ser?Ser?Arg?Leu?Gln?Asp?Leu?Tyr?Ser?Ile?Val?Arg?Arg?Ala

50 55 60Asp?Arg?Ala?Ala?Val?Pro?Ile?Val?Asn?Leu?Lys?Asp?Glu?Leu?Leu?Phe65 70 75 80Pro?Ser?Trp?Glu?Ala?Leu?Phe?Ser?Gly?Ser?Glu?Gly?Pro?Leu?Lys?Pro

85 90 95Gly?Ala?Arg?Ile?Phe?Ser?Phe?Asp?Gly?Lys?Asp?Val?Leu?Arg?His?Pro

100 105 110Thr?Trp?Pro?Gln?Lys?Ser?Val?Trp?His?Gly?Ser?Asp?Pro?Asn?Gly?Arg

115 120 125Arg?Leu?Thr?Glu?Ser?Tyr?Cys?Glu?Thr?Trp?Arg?Thr?Glu?Ala?Pro?Ser

130 135 140Ala?Thr?Gly?Gln?Ala?Ser?Ser?Leu?Leu?Gly?Gly?Arg?Leu?Leu?Gly?Gln145 150 155 160Ser?Ala?Ala?Ser?Cys?His?His?Ala?Tyr?Ile?Leu?Leu?Cys?Ile?Glu?Asn

165 170 175Ser?Phe?Met?Thr?Ala?Ser?Lys

180

Claims (16)

1. a method for preparing chalone in the recombinant human is characterized in that using and contains 1 at N end, and the colibacillus engineering strain of the high expression level of chalone gene is carried out in the people of the coding nonsense mutation of 3-5 amino acids.

2. according to 1 at the N end that the process of claim 1 wherein, the chalone gene is the nucleic acid shown in the SEQ ID No.3 (accompanying drawing 3) in the people of the coding nonsense mutation of 3-5 amino acids

3. according to the method for claim 1 or 2, wherein utilize lactose, induce chalone efficiently expressing in intestinal bacteria in the recombinant human as inductor.

4. according to the method for claim 3, wherein lactose-induced is by growing to OD at the bacterium engineering strain ₆₀₀During 14-18, add lactose and carry out.

5. according to the method for claim 4, wherein the ultimate density of the lactose of Jia Ruing is 2%.

6. according to the method for claim 1 or 2, wherein utilize the leakage of glucose control protein expression.

7. according to the method for claim 6, the leakage of protein expression wherein is by before abduction delivering not, adds glucose and carry out in substratum.

8. according to the method for claim 1 or 2, colibacillus engineering strain wherein is that preserving number is intestinal bacteria BL-21 (DE-3)/pENDO of CCTCC NO:M203063.

9. the expression vector that one kind big enterobacteria engineering strain, this bacterial strain have been contained the nucleic acid shown in the SEQ ID No.3 (perhaps accompanying drawing 3) transforms.

10. according to the big enterobacteria engineering strain of claim 9, it is to be preserved in Chinese typical culture collection center (CCTCC, Wuhan) and preserving number is intestinal bacteria BL-21 (DE-3)/pENDO of CCTCC NO:M203063 on August 6th, 2003.

11. chalone in the recombinant human, it is to be produced by each method among the claim 1-8.

12. a nucleotide sequence, it is as SEQ ID No 3 and shown in Figure 3.

13. the nucleotides sequence of claim 12 is listed in purposes in the colibacillus engineering strain that makes up chalone in the efficiently expressing recombinant human.

14. lactose-induced method is the purposes in the efficiently expressing of chalone in inducing recombinant human.

15. chalone is used for the purposes of the medicine of anti-neovascularization in the recombinant human that each method is produced among the claim 1-8 in preparation.

16. the purposes of claim 15, medicine wherein is used for antitumor.

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