CN1050862C

CN1050862C - Method for human urokinase and other useful proteins efficiently demonstrated by insect cells

Info

Publication number: CN1050862C
Application number: CN93102115A
Authority: CN
Inventors: 徐扬; 胡美浩; 张洪涛
Original assignee: Peking University
Current assignee: Peking University
Priority date: 1993-03-04
Filing date: 1993-03-04
Publication date: 2000-03-29
Anticipated expiration: 2013-03-04
Also published as: CN1075165A

Abstract

The present invention constitutes a novel high efficiency transferring carrier for expressing insect nucleus multi-plasmid embedding polyhedral viruses by means of a gene engineering technology. The gene of a plasmid polyhedral protein promoter has a complete structure, and a 3' terminal homological sequence area is smaller than a common transferring carrier plasmid. Human pro-urokinase genes are inserted into the plasmid to obtain high efficiency expression. By means of homological recombination of cultured insect cells, recombinant insect nucleus multi-plasmid embedding polyhedral viruses containing the human pro-urokinase genes are obtained. Therefore, the infected insect cells express human pro-urokinase with high activity and high efficiency and are directly secreted into the supernate of a culture medium. Expressed human pro-urokinase protein is recovered and purified by a single process of monoclonal antibody affinity chromatography.

Description

Utilize insect cell to efficiently express human pro-urokinase and other method of useful proteins

Utilize insect cell to efficiently express the human pro-urokinase and other method of useful proteins belongs to the biological gene engineering field, the present invention relates to produce human pro-urokinase and other method of useful proteins.Thereby the prognosis that present cardiovascular and cerebrovascular thrombus complication has a strong impact on clinical patients myocardial infarction, cerebral thrombosis causes high mortality.Therefore, attempt using the thrombolytic therapy thrombus both at home and abroad in recent years, the clinical report of control cardiovascular and cerebrovascular thrombus complication is more and more.After particularly entering the 80s and 90s, thrombolytic therapy becomes the routine treatment of Acute Myocardial Infarction; The used thrombolytic drug of thrombolytic therapy is plasminogen activator (plasminogen activator, be abbreviated as PA), single chain urokinase type plasminogen activator type PA is human pro-urokinase (prourokinase, be abbreviated as pro-UK) be a kind of novel thrombolytic agent, be better than urokinase (urokinase, be abbreviated as UK), have fibrinogen (thrombus) effect specificity.But pro-UK does not resemble and is easy to prepare the UK, has only genetically engineered to produce and could solve this difficult problem.

Microflora such as intestinal bacteria, yeast has been used to express outer protogene and has produced, and having formed some gene engineering products but because colibacillary expression product forms a kind of undissolved inclusion body needs by denaturing agent dissolving and renaturation with the recovery natural structure, the biological activity that makes pro-UK one class have many protein expression products to disulfide linkage is greatly affected.The expression product of mammalian cell keeps natural three-dimensional structure, can be with the protein side chain glycosylation, and posttranslational modification processing and secretion, but the mammalian cell cultivation costs an arm and a leg, and strictness of equipment and technology conditional request and expression efficiency are not high.People attempt with insect cell as the expression of exogenous gene system in recent years, being characterized in not only can the monolayer adherence growth but also suspension culture in a large number, do not contain still well-grown of foetal calf serum cell in the substratum, have the glycosylation of expression product side chain, post-treatment modification and secreting function.Use the tens kinds of foreign genes that have of this system expression, mostly be virus capsid protein, for example:

1. wear dimension HL Bi Xiaopu, health is grant forever: patent application CN 87106266A, denomination of invention: the expression of hepatitis B virus antigen in the recombinant baculovirus carrier, on September 8th, 1987.This invention is to express human body hepatitis B virus antigen and the pre-S2 albumen relevant with hepatitis B surface antigen(HBsAg) with insect expression system.Similarly reta virus and vaccine thereof, japanese encephalitis virus, canine parvovius vaccine, HIV (human immunodeficiency virus) are for example expressed in invention, see European patent, Japanese Patent etc.

2.Summers，M.D?etal：United?Staes?Pateat?5023328，Title：Lepidopteran?AKH?signal?sequeace，Jun.11，1991。This invention is the lipotropin of lepidopterous insects to be secreted peptide dna encoding sequence be inserted into metastasis transplanting physique grain polyhedrin promotor downstream, and making the foreign gene of not being with the secretion peptide-coding sequence express justacrine at insect expression system becomes possibility.

3.Wang, N.P.etal:Cell Growfh ﹠amp; Differentiation.1990,1:429-437 described in the literary composition, adopts insect expression system to express reticulocyte cell tumor expression product pp110 ^RBProtein is about 10mg/liter substratum supernatant.Similarly expression product has G CFS, and the sweet enzyme A of α-half hole sugar, growth factor of human nerve etc. see world patent, European patent etc.But its general character is that expression output is not high enough.Be that expression product possesses natural character with active, but expression efficiency is not high enough, is unfavorable for forming gene engineering product.The high not most critical of expression efficiency be the structure of the metastasis transplanting physique grain that is used for construction of recombinant virus, inserts for foreign gene, the polyhedrin promoter gene is more complete in the metastasis transplanting physique grain, expression efficiency is higher.

The objective of the invention is to obtain a kind of (2) and efficiently express human urokinase crude protein and other useful proteins gene engineering method by making up better (1) metastasis transplanting physique grain.The human pro-urokinase is a kind of novel thrombolytic agent, but is difficult to obtain from nature, has only genetically engineered to produce and could solve this difficult problem.Expression efficiency of the present invention is far above the expression amount of mammalian cell culture supernatant, and is better than intestinal bacteria and produces expression product (the sex change renaturation greatly reduces the biological activity that reclaims the human pro-urokinase), is with a wide range of applications.

Content of the present invention and scheme: make up the metastasis transplanting physique grain pAcYT that efficiently expresses, the human cloning pro-urokinase cDNA is to metastasis transplanting physique grain.The metastasis transplanting physique grain and many embedding type polyhedrosis virus DNA cotransfection insect Sf cells of wild-type nuclear that will have human urokinase protogene's (containing the secretion peptide-coding sequence).Plaque method screening purification of Recombinant virus.Human urokinase crude protein with recombinant virus infection insect Sf cell harvesting secreting, expressing.Present method is suitable for utilizing expresses other useful proteins matter with quadrat method.

Embodiment:

1. make up the metastasis transplanting physique grain pAcYT efficiently express: with digestion with restriction enzyme remove plasmid pAc373 promoter gene the XhoI-BamHI fragment (2,1Kb), do not contained the big fragment of pAc373 ' DNA XhoI-BamHI (7.7Kb) of promotor.With 3 '-(the XhoI-BamHI fragment 2.1Kb) is connected with the big fragment of pAc373 ' DNA XhoI-BamHI of 7.7Kb for the clone wild-type AcMNPV DNA polyhedrin promoter gene fragment of downstream modified.Remove the dna fragmentation (BamHI-KpaI fragment) in one section polyhedrin structural gene downstream again, obtain a new promotor downstream DNA fragment-7TATAAAT-1 and lack the segmental transfer vector pAcYT that efficiently expresses of polyhedrin structural gene downstream BamHI-KpnI with interpolation.Determined dna sequence shows: the pAcYT promoter gene is Duoed one section TATAAAT sequence than pAc373.Be the present invention constructed to efficiently express transfer vector pAcYT promotor part-structure more complete than pAc373, foreign gene is expressed under complete polyhedrin promotor control.

2. the human cloning pro-urokinase cDNA is to transfer vector pAcYT: segmentation recombinant human urokinase zymogen cDNA is last to obtain BamHI, KpnI end to plasmid Blae-script and pUC19 respectively through secondary, then the tack forward be inserted on the BamHI site of metastasis transplanting physique grain pAcYT (see figure 1) with the pAcYT/UK plasmid amplification after, carry out purifying with conventional DNA purification process such as cesium chloride density gradient centrifugation or PBG8000 precipitations.

3. virus and cell and viral DNA preparation: insect cell line Sf9 cell is cultivated in TMN-FH or IPL41 substratum: insect cell line Sf21 cell is cultivated in the TC-100 substratum; Infect the Sf cell with virus of proliferation with many embedding type polyhedrosis viruses of nuclear.Virus particle in high speed centrifugation (4 ℃, 30000 rev/mins) the precipitation substratum supernatant, lower concentration Na ₂CO ₃The cracking polyhedron, Proteinase K hydrolysis virus protein, the extracting of phenol chloroform once, the ethanol sedimentation viral DNA.

4. the screening of homologous recombination virus in the insect cell: the pAcYT/UK DNA 1-5 microgram and the wild-type AcMNPV DNA2-10 microgram that will have human pro-urokinase cDNA (containing the secretion peptide-coding sequence) are suspended in the 30 microlitre distilled water, mix back cotransfection monolayer adherence Sf cell with 30-50 microgram liposome, 25 ℃-28 ℃ incubation 2-4 hour.Transfection liquid is abandoned in suction, renews bright substratum, cultivates 4-7 days for 25 ℃-28 ℃, forms until viral polyhedron.There is not polyhedron (Occ according to recombinant virus ^-) this morphological feature, with plaque method screening purifying, triplicate.Obtain recombinant virus.

5. the recombinant virus infection Sf9 cell that has the human urokinase protogene: every milliliter of Sf9 cell is with 10 ³The recombinant virus infection of PFU (plaque forming unit) is collected substratum supernatant (containing the expression product human pro-urokinase) after 4-7 days, and is contrast with the wild virus.

Purifying with reclaim expression product human urokinase crude protein: will adorn post after the Sepharose 4B gel of cyanogen bromide-activated and anti-human urokinase monoclonal antibody (4D1E8) coupling, 2.5 milliliters of column volumes are gone up sample volume 30-60 milliliter at every turn.Use the 2mol/LKSCN wash-out, collector's uPA elution peak effluent liquid, saturating folding in the presence of Aprotinin (Sigma), low-temperature vacuum drying.The BLISA method is determined the human urokinase original content, and the standard substance human pro-urokinase is the GmbH product, and concentration is accurately quantitative through the aminoacid component analysis.Fibrinogen plate assay is measured human pro-urokinase's fibrinolytic biological activity.Human pro-urokinase's expression product of SDS-polyacrylamide gel electrophoresis and Weatera blot purification Identification.Recombinant virus infection Sf cell is after 5-7 days, the human pro-urokinase is 96mg/liter (a TMN-FH substratum) in the supernatant liquor, 60mg/liter (Sf900 serum free medium), 16mg/liter (IPL11 substratum), expression amount is also different according to the substratum difference, and cellulolytic activity is 800000IU～1600000IU/liter.Monoclonal antibody affinity chromatography single stage method purifying expression product, the rate of recovery reaches more than 70%, is about 60000IU/mg than living; The human urokinase crude protein of purifying, no matter non-reduced still reduction is handled, and its SDS-polyacrylamide gel electrophoresis collection of illustrative plates is identical list and uses band, and molecular weight is about 50000; Its West-era blot result is single band, coincide with SDS-polyacrylamide gel electrophoresis collection of illustrative plates.

Description of drawings:

Fig. 1 metastasis transplanting physique grain pAcYT polyhedrin promoter gene 3 '-the end dna sequence dna

Measure collection of illustrative plates

Claims

1. one kind is utilized insect to efficiently express human pro-urokinase and other method of useful proteins, it is characterized in that efficiently expressing human pro-urokinase and other useful proteins matter with the insect cell that recombinant baculovirus infects, and described method comprises:

(1) makes up the metastasis transplanting physique grain PAcYT that efficiently expresses:, do not contained the big fragment 7.7Kb of pAc373 ' DNA XhoI-BamHI of promotor with the XhoI-BamHI fragment 2.1Kb of digestion with restriction enzyme removal plasmid pAc373 promoter gene; With 3 '-the clone wild-type AcMNPV DNA polyhedrin promoter gene fragment XhoI-BamHI fragment of downstream modified, 2.1Kb is connected with the big fragment of pAc373 ' DNA XhoI-BamHI of 7.7Kb; In the dna fragmentation BamHI-KpnI fragment of removing one section polyhedrin structural gene downstream, obtain a new promotor downstream DNA fragment TATAAAT with interpolation, and the disappearance polyhedrin structural gene downstream BamHI-KpnI segmental transfer vector pAcYT that efficiently expresses, determined dna sequence shows: the pAcYT promoter gene is Duoed one section TATAAAT sequence than pAc373 and is seen Fig. 1; Be the present invention constructed to efficiently express transfer vector PAcYT promotor part-structure more complete than pAc373, foreign gene is expressed under complete polyhedrin promotor control;

(2) the human cloning pro-urokinase cDNA is to transfer vector pAcYT: through secondary respectively segmentation recombinant human urokinase zymogen cDNA go up to obtain BamHI, KpnI end to plasmid Blue-script and pUC19, the tack forward is inserted on the BamHI site of metastasis transplanting physique grain pACYT and sees Fig. 1 then; Behind the pAcYT/uK plasmid amplification, carry out purifying with conventional DNA purification process such as cesium chloride density gradient centrifugation or PEG8000 precipitations;

(3) virus and cell and viral DNA preparation: insect cell line Sf9 cell is cultivated in TMN-FH or IPL41 substratum; Insect cell line Sf21 cell is cultivated in the TC-100 substratum; Infect the Sf cell with virus of proliferation with many embedding type polyhedrosis viruses of nuclear; 4 ℃ of high speed centrifugations, 30000 rev/mins of virus particle that precipitate in the substratum supernatant, lower concentration Na ₂CO ₃The cracking polyhedron, Proteinase K hydrolysis virus protein, the extracting of phenol chloroform once, the ethanol sedimentation viral DNA;

(4) screening of homologous recombination virus in the insect cell: will have human pro-urokinase cDNA and contain the pAcYT/uK DNA 1-5 microgram and the wild-type AcMNPVDNA 2-10 microgram of secreting peptide-coding sequence and be suspended in the 30 microlitre distilled water, mix back cotransfection monolayer adherence Sf cell with 30-50 microgram liposome, 25 ℃-28 ℃ incubation 2-4 hour.Transfection liquid is abandoned in suction, renews bright substratum, cultivates 4-7 days for 25 ℃-28 ℃, forms until viral polyhedron.There is not polyhedron Occ according to recombinant virus ^-This morphological feature, with plaque method screening purifying, triplicate obtains recombinant virus;

(5) have human urokinase protogene's recombinant virus infection Sf9 cell: every milliliter of Sf9 cell is with 10 ³The recombinant virus infection of PFU is collected the substratum supernatant and is contained the expression product human pro-urokinase, and is contrast with the wild virus after 4-7 days;

(6) purifying with reclaim expression product human urokinase crude protein: will adorn post, 2.5 milliliters of column volumes after the Sepharose 4B gel of cyanogen bromide-activated and the anti-human urokinase monoclonal antibody 4D1E8 coupling; Each sample volume 30-60 milliliter of going up; Use the 2mol/LKSCN wash-out, collector's uPA elution peak effluent liquid is rolled in the presence of Aprotinin thoroughly, low-temperature vacuum drying,

2. method according to claim 1, wherein the metastasis transplanting physique grain PAcYT that efficiently expresses of the structure in the step (1) is characterized in that the polyhedrin promotor polyhedrin promoter that said viral metastasis transplanting physique grain promotor is a nucleopolyhedrosis virus;

3. method according to claim 1, the cell in the step (3) wherein is characterized in that it is a kind of insect cell greedy frugiperda cell Spodopterafrugiperda in order insect meadow that promptly derives, and is abbreviated as the Sf cell;

4. method according to claim 1, the virus in the step (3) wherein, it is characterized in that it be a kind of insect viruses promptly: many embedding type polyhedrosis virus Autogapha CaliforrnicaNuclear Polyhedrosis Virus of autographa california nuclear are abbreviated as AcMNPV;

5. method according to claim 1, wherein the human urokinase protogene in the step (5) is characterized in that the human pro-urokinase comprises that human urokinase protogene or its part are from the corresponding base of 144 amino acids residues initial part A chain and B chain gene or other portion genes;

6. method according to claim 1, wherein said and other useful proteins matter are meant that the protein of various useful biologically actives comprises and contain sugar-protein, contain disulfide bond protein matter and some specific proteins.