CN1058752C - Making-up high effective expression vector using E. coil intensified subsample sequence and application thereof - Google Patents

Abstract

A colibacillus enhancer detection carrier is used for cloning an 894 bp segment (called an M segment) with the function of enhancer samples fromcolibacillus chromosomal DNA, and simultaneously proving a segment (called an X segment) about 300 bp in the upstream control region of a PL starter, an SV40HindIIIB segment, a vaccinia virus HindIIIK segment and an RSVG region. The URR regions, etc. of HPV6B and HPV16 all have obvious enhancer effect. Two kinds of expression carriers are constructed by the M segment and the X segment. The two kinds of expression carriers have obvious enhancer sample effects on the expression of multiple exogenous genes, and are capable of enhancing the yield of recombination polypeptide medicines of colibacillus systems by 3 to 9 times.

Description

Adopt intestinal bacteria to strengthen increment sequence construct efficient expression vector and uses thereof

The present invention relates to the enhanser field of gene transcription regulation in the genetic engineering.

Genetic transcription is regulated and is made up of two class sequential elements basically: the one, and promoter element, the 2nd, regulon element.Promoter element is essential to accurate, effective transcription initiation, and the regulon element then works to strengthen or weaken promoter transcription efficient.The regulon element generally is positioned at mRNA transcription initiation upstream, at least more than the 100bp at the far-end of specific gene transcription initiation.What study more detailedly is enhancer sequence.Typical enhanser is 70～150bp, can with specific protein-interacting, strengthen and transcribe efficient.Enhanser often is made up of one or more continuous or discrete dna sequence dna elements.

Since 1981 (1,2) such as Banerji find that there is enhancer sequence in the SV40 genome, found that in succession this genetic transcription adjusting sequence extensively is present in eukaryotic cell and the viral genome thereof.Think that at present enhanser is an important and essential element of eukaryotic gene transcriptional control.

(3) such as Harley had analyzed intestinal bacteria in 1987, comprised 263 promoter sequences of its plasmid transposon and lambda particles phage, thought that still-35 (TTGACA) and-10 (TATAAT) are high conservatives.92% promotor is arranged, and the distance of two conserved regions is 17 ± 1bp.This class prokaryotic promoter can be and contains the RNA polymerase of " normally " Sigma 70 factors and discerns, and is called " Pribnow " type promotor again.An other class prokaryotic promoter is called Ntr type promotor, it does not have-10 and-35 conserved regions and have-12 and-24 conserved regions, it can be the RNA polymerase that contains Sigma 60 factors and discerns, and this class promotor comprises intestinal bacteria glnAP2, the nif of pneumobacillus etc.(4) such as Nishi thought that the upstream A+T of intestinal bacteria trp and PL promotor enriched the district and can strengthen its transcriptional activity in 1986.1986 (5) such as Buch find that the upstream of nif promotor has the increment of enhancing sequence.1987 (6) such as Garciarrabio discovery has the increment of enhancing sequence in intestinal bacteria glnA upstream region of gene district.(7,8) such as Huber in 1985 and Kahmann find the G sections of Mu phage, and there is enhancer sequence in the H sections of salmonella typhi in the site-specific sex-reversal recombination system of the C sections of P1, P7 phage etc.(9) such as nineteen ninety Latta prove that trp promotor upstream AT enriches the district and can make gene expression dose increase by 2 times.To bring into play its function completely no matter this explanation is " Pribnow " type or " Ntr " type promotor, also should comprise the upstream regulation district of promotor.

Present inventors found in research people α, interferon-gene accident when colibacillary expression saves that SV40 Hind IIIB fragment was in the expression that can strengthen Interferon, rabbit cDNA at a distance of promotor 1000pb place significantly in 1984.Subsequently, present inventors find HPV-6BURR in succession, HPV-16URR, vaccinia virus HindIIIK fragment and RSVG fragment all have similar enhanser effect, and the characteristics of the pronucleus enhancer sample sequence of this class animal-origin (V-EE) are: 1. 0.3～0.9Kb place in promotor upstream can make the expression level of reporter gene improve 3-10 doubly; 2. its reinforcing effect does not have tangible directivity; 3. strengthen increment sequence itself and also have promoter function simultaneously, but the sequence with promoter function not necessarily has the increment of enhancing effect; 4. some pronucleus enhancer sample sequence can work to escherichia coli promoter, also can work to the bovine vaccine promotor.

Present inventors have been set up intestinal bacteria and strengthen to have been detected carrier, the new detection carrier of setting up comprise following characteristics: a. have a more weak escherichia coli promoter sequence b. have in the promotor downstream reporter gene that is easy to detect (CAT.lac, IFN); C. a multienzyme point of contact is arranged more than the 300bp of promoter sequence upstream and whether have the increment of enhancing activity so that detect multiple sequence.Directly from e. coli chromosomal dna the clone heavy one have the about 894bp fragment that strengthens the increment function, name fragment for M, and measured its complete sequence, adopt Prosis and DNAsis software and Gene BANK and EMBL DNA and protein pool to carry out sequence homology relatively, find that it is intestinal bacteria (JM103) coding methionine(Met) (Methionine amio peptid) and 30s Ribosomal proteinS2 (PPSB) portion gene and upstream regulatory region thereof that M strengthens the increment sequence.Disappearance studies show that the segmental functional site of M enhanser sample is distributed in the 450-800bp scope of forward clone 3 ' end upstream.

Transcribe test by body is inside and outside, Northern hybridization, further proof M fragment to truly had the enhancement on transcriptional level by regulatory gene, proves also that simultaneously to have several DNA that can be incorporated into the M sequence in intestinal bacteria conjugated protein in Bacillus coli cells.

Also further proof PRPL promotor and upstream regulatory region (300bp) (X fragment) thereof place trp promotor upstream (300bp) to locate to present inventors, the trp promotor also there is tangible enhancing increment effect, under the condition that clts 857 exists, the segmental reinforcing effect of X is not subjected to the influence of temperature variation (30-42 ℃).Dna sequence dna homology analysis comparative result shows that also this class has the animal virus gene fragment of pronucleus enhancer sample function: the URR district of SV40 HindIII B fragment, HPV6B and HPV16, RSVG district, vaccinia virus HindIII K fragment etc. and a plurality of following conserved sequences: TGTNxACA is all arranged from intestinal bacteria isolated M fragment.This conserved sequence also is common in the transcriptional regulatory district of nif.After reaching simultaneously, just be reported in succession in the world in the reverse system with present inventors, the glnA gene, the nifH gene of pneumobacillus, and intestinal bacteria trp gene etc. all confirms to have enhancer sequence.

On the basis of above-mentioned research, present inventors adopt intestinal bacteria to strengthen the increment sequence, have made up two class expression vectors, pBV321/322 and pBV324, the former is with the X fragment, and the latter is with the M fragment, proves the β to IFN-, IFN-α 2a, IFN-α 2b, IFN-α 1b/86C, FN-α 1 b/86D, I FN-γ, the isogenic expression of TNF-α all has tangible enhancement.

The objective of the invention is to determine that also there is enhancing increment sequence in the intestinal bacteria system, and separate the intestinal bacteria enhanser make new advances, an artificial constructed thus class adopts intestinal bacteria to strengthen the efficient carrier of increment sequence, thereby improves the output of recombinant polypeptide medicine greatly.

In order to realize purpose of the present invention, the present invention is by the following technical solutions: the present inventor has made up the intestinal bacteria enhanser and has detected carrier, utilize the equimolecular biology techniques from e. coli chromosomal dna, to clone one and have the 894bp fragment (claiming the M fragment) that strengthens the increment function, proved that all there is tangible enhancing increment effect in the upstream regulatory region (claiming the X fragment) of about 300bp of PL promotor and the URR district of vaccinia virus HindIII K fragment, RSV G district, HPV6B and HPV16 etc. simultaneously again.The present inventor utilizes M fragment and X fragment to make up two class expression vectors simultaneously, pBV321/322 and pBV324, and the former has the X fragment, and the latter is with the M fragment.

The present invention compared with prior art has following beneficial effect: at first, the present invention has proved the multiple fragment that intestinal bacteria strengthen the increment sequence that has, and these fragments are used to make up coli expression carrier, can improve the output of recombinant polypeptide medicine.Secondly, utilize M fragment and X fragment to make up two class expression vectors.Utilize this two classes expression vector to produce the recombinant polypeptide medicine, can improve output 3-9 doubly.

Explain the present invention below in conjunction with accompanying drawing, wherein:

Fig. 1 is a SV40HindIII B fragments sequence;

Fig. 2 is a vaccinia virus Tiantan strain genome HindIIK/BglII2.2kb fragments sequence;

Fig. 3 is a RSVcDNABamHI G fragment sequence;

Fig. 4 is the 540bp sequence of HPV-6B type;

Fig. 5 is a HPV-16 type 7013-7453bp sequence;

Fig. 6 strengthens the increment sequence for intestinal bacteria M;

Fig. 7 is a λ PRPL promotor upstream regulatory sequence;

Fig. 8 is the plasmid figure of pBV320;

Fig. 9 is the plasmid figure of pBV321;

Figure 10 is the plasmid figure of pBV324;

Also the invention will be further described to explain technical scheme of the present invention below in conjunction with embodiment.The following example only is used to explain the present invention, the scope of the invention is not construed as limiting.

Implementing 1. is the establishment that body is cut out in the detection of reporter gene with CAT

Escherichia coli plasmid pkk232-8, available from Phamacia company, it has a promoterless chloramphenicol acetyltransferase (ChloramphenicolAcetyltransferase is called for short CAT) gene.In CAT gene 3 ' end downstream two place's transcription termination signales are arranged, to prevent transcribing and duplicating of strong promoter interference plasmid.At 5 of CAT gene ' end one multienzyme joint is arranged, also there is place's transcription termination signal its upstream, with the effect of the promotor of avoiding deriving from upstream pBR322.Between the ATG of multienzyme joint and CAT gene, the translation termination signal of 3 different single open reading frames is arranged, to avoid possible genetic expression from the upstream.With this plasmid is parent material, makes up the enhancer sequence with multienzyme point of contact-vaccinia virus promotor-CAT respectively and detects carrier, the wherein distance＞300bp between multienzyme point of contact and the promoter sequence.The enhancing increment sequential detection carrier that has vaccinia virus 7.5K promotor claims pTE 7.5; The enhancing increment sequential detection carrier that has vaccinia virus 11K promotor claims pTE11; The title pKN2 that has vaccinia virus N promotor.

Embodiment 2. is the establishment of the detection carrier of reporter gene with lac

With beta-galactosidase gene (lac) is reporter gene, and the enhancing increment sequential detection carrier of structure claims pAL and pLW.Its sequence in the gene is 5 ' multienzyme point of contact-300bp-p7.5 promotor-beta-galactosidase gene, and pAC is the different of multienzyme point of contact with the difference of pLW.

Embodiment 3. is the establishment of the detection carrier of reporter gene with the human interferon

Adopt the pKC-30 plasmid earlier, insert people α I type Interferon, rabbit cDNA in the HpaI site, express under the control of PL promotor, there is the HindIII site at the 1000bp place in distance PL promotor upstream, strengthens the increment sequence to detect.The detection carrier of setting up claims pKC-30/IFN-α 1.

The genomic intestinal bacteria of embodiment 4.SV40 strengthen determining of increment sequence

With the HindIII site that the SV40HindIIIB fragment of about 1kb is inserted the PKC30/IFN-a11 carrier, can make the expression amount of reporter gene increase by 6 times.The segmental complete sequence of SV40HindIII B is seen accompanying drawing 1

The segmental intestinal bacteria of embodiment 5. vaccinia virus HindIIIK strengthen determining of increment sequence

Adopt Lac to detect the BglII fragment of the 2.2kb in the carrier proof vaccinia virus Tiantan strain HindIIIK fragment, the expression of reporter gene is had tangible enhancement.When the host bacterium was JM103, when the BglII fragment of no HindIII K was inserted, the expression amount of Lac was 285 ± 37 units, and forward is 1387 ± 268 units when inserting, and is 437 ± 48 units when oppositely inserting.When the host bacterium is MC1061, respectively be 169 ± 38 units, 558 ± 116 units and 261 ± 45 units.The segmental DNA complete sequence of vaccinia virus Tiantan strain HindIIIK is seen accompanying drawing 2

The intestinal bacteria of the BamHI G fragment (0.7kb) of embodiment 6. respiratory syncytial virus (RSV) cDNA strengthen determining of increment sequence

The BamHI G fragment of employing Lac detection carrier proof RSV cDNV has tangible enhancement to the expression of reporter gene, when the host bacterium is JM103, when not having the fragment of insertion is 2.9 ± 0.9 units, and forward is 21.2 ± 2.0 units when inserting, and is 21.9 ± 1.2 units when oppositely inserting.Respiratory syncytial virus (RSV) cDNA BamHI G fragment complete sequence is seen accompanying drawing 3

The intestinal bacteria of embodiment 7. human papillomavirus 6B type genome URR sections strengthen determining of increment sequence

Downcut the full genome of HPV6b with BamHI from the pBR322 plasmid, be digested to 19 fragments with Hae III again, shotgun cloning is to above-mentioned enhancing increment sequential detection carrier S ma I site, and transformant is containing the enterprising row filter of plate of paraxin 500 μ g.(not having the segmental pTE 7.5 of insertion can not grow on the plate of the plain concentration of this chlorine enzyme).After the bacterium colony of growth extracted plasmid, cut through enzyme and to examine and determine these positive colonies with molecular hybridization and all contain HaeIII 2.3kb fragment (pTEL1), this is equivalent to HPV-6B URR district.The chlorampenicol resistant strength detection represents that transformant can grow under the concentration of 600 μ g/ml, promptly do not increased about 5 times than there being the resistance of inserting the fragment plasmid.Change this fragment over to lac and detect carrier, when the host bacterium was JM103, contrasting no insertion group Lac activity was 10.4 ± 2.8, and forward is inserted as 39.3 ± 2.2, oppositely is inserted as 21.6 ± 2.2 units/ml.

Be the accurate position of research performance enhanser effect, present inventors carry out the segmental deletion analytical test of HaeIII2.3kb.Progressively delete test with the method that enzyme is cut from the promotor of band EI gene and HPV6B.Obtain the fragment of 540bp at last, and carried out dna sequencing, the result shows: just have near the 540bp sequence of E6 gene A TG upstream and strengthen the increment function completely, betagalactosidase activity is measured and is shown, when the host bacterium is JM103, the Lac activity that contrasts no insertion group is 10.4 ± 2.8, and forward is inserted as 108.9 ± 16.1, oppositely is inserted as 30.6 ± 2.1 units/ml; When the host bacterium was MC1061, control group was 8.5 ± 0.3; Forward is inserted as 46.0 ± 4.4; Oppositely be inserted as 21.0 ± 8.8 units/ml.

Equally the URR fragment cloning of 540bp is carried out the mensuration of chlorampenicol resistant to the multiple clone site of pKN2, the result observes the expression of gene to CAT, and enhancement is also arranged.The dna sequence dna in 540bp-HPV6B URR district is seen accompanying drawing 4

The intestinal bacteria of embodiment 8. HPV 16 genome URR sections strengthen determining of increment sequence

Present inventors select pAL to detect in the multienzyme joint of carrier promotor upstream the BglII site as cloning site.HPV16 genome 1.4Kb Sau3AI endonuclease bamhi is inserted this site, obtain the recombinant plasmid (pAL01 and pAL02) of two kinds of directions.Present inventors are with two kinds of recombinant plasmid transformed intestinal bacteria JM103 bacterial strains.Choose single bacterium colony activation culture and carry out betagalactosidase activity mensuration, the result shows that the Lac activity that contrasts no insertion group is 5.4 ± 1.0; Forward is inserted as 14.1 ± 1.1; Oppositely be inserted as 22.4 ± 1.2 units/mL.

Further more accurate intestinal bacteria strengthen the increment active region in research HPV16 genome middle and upper reaches regulatory region 1.4Kb (14U) fragment.Present inventors have dwindled the 14U fragment to a greater degree.The enhanced activity sequence of finding the trip regulatory region mainly concentrates in the EcoRI enzymolysis small segment of 14U, is present in the full genome 7013-7453bp of the HPV-16 that has reported position, and length overall is 440bp.Present inventors are referred to as 44U with it.In order further to prove the enhancing increment activity of this section sequence, present inventors separate big fragment with pAl01 plasmid EcoRI enzymolysis through agarose gel electrophoresis, connect with ligase enzyme again, obtain plasmid pAL011.With HindIII and SphI enzymolysis pAL02 plasmid, separate big fragment through agarose gel electrophoresis, scabble sticky end with nuclease SI again, connect with ligase enzyme then, obtain plasmid pAL022.These two kinds of plasmid clones' fragment is the insertion of same fragment different directions.Two kinds of plasmids are transformed the JM103 bacterium simultaneously, detect wherein betagalactosidase activity.The result shows that control group Lac activity is 1.8 ± 0.3; Forward is inserted as 11.8 ± 1.8; Oppositely inserting the people is 7.2 ± 1.0 units/mL.This enhancer sequence is seen accompanying drawing 5

Embodiment 9. intestinal bacteria strengthen determining of increment sequence

Adopt the pKN2 plasmid as the detection carrier that strengthens the increment sequence, the CAT gene has and can the resistance of the paraxin of different concns be evaluated by detecting its host bacterium according to CAT expression of gene level in the vaccinia virus N promotor of intestinal bacteria work in the plasmid.The chlorampenicol resistant of pKN2 is 100-200 g/ml.Intestinal bacteria JM103 chromosomal DNA is handled with the bean sprouts nuclease with restriction enzyme HindIII and HaeIII digestion.The big or small fragment of each that is obtained is cloned in the SmaI site of pKN2, the high concentration cl mycin that transformed bacteria can not be grown at former plasmid pKN2 former bacterium (400 μ g) agar plate carries out the pressure screening, the result selects a strain, and to insert clip size be positive colony bacterium pM1.6 about 0.9kb, through cross experiment, prove that this fragment derives from intestinal bacteria JM103 chromosomal DNA.The chlorampenicol resistant of measuring pM1.6 is 600-800 μ g/ml, can make chlorampenicol resistant improve 4-5 doubly.Present inventors claim that this cloned sequence is JM103-M (being called for short the M fragment).

The JM103-M fragment not only can make its contiguous CAT genetic expression that tangible reinforcing effect is arranged, and, beta-galactosidase enzymes is also had similar effect.Selecting pG7 (-) is cloning vector.With HindIII and EcoRI digestion pM1.6, take out the JM103-M fragment, digest with HindIII after pG7 (-) DNA that the parlkaline Phosphoric acid esterase is handled is connected with elder generation, again the DNA that connects is handled the unmatched strand end of removal with the bean sprouts nuclease, be connected with the T4 dna ligase.Selecting especially blue conversion bacterium colony on the X-gal of 20ug/ml plate extracts plasmid and carries out enzyme and cut evaluation, the result has obtained different two the recombinant plasmid pGM (-) of JM103-M fragment direction of insertion, pGM (+), present inventors are to pGM (-), pGM (+), the betagalactosidase activity of pG7 (-) in JM103 host bacterium compares, found that, the JM103-M fragment is inserted pG7 (-) with two kinds of directions, all the expression to beta-galactosidase gene has tangible reinforcing effect, wherein, identical person [pGM (+)] is for the highest among the segmental direction of insertion of M and the pM1.6, direction opposite [pGM (-)] takes second place, and learning by statistics to handle has, both differences of no JM103-M all very remarkable (P＜0.01).The segmental complete sequence of M is seen accompanying drawing 6.

Embodiment 10. profits use the same method and have determined that PRPL promotor upstream regulatory region (X fragment) sees accompanying drawing 7 for the complete sequence that intestinal bacteria strengthen increment sequence PRPL promotor upstream regulatory regions (X fragment).

The function of embodiment 11. above-mentioned enhancing increment sequences is summed up as table 1.

Table 1 strengthens the function of increment sequence

The sweet enzymic activity of beta galactose increases multiple

Sequence source host

Do not have the forward of insertion and insert oppositely insertion V-K bovine vaccine JM103 4.9 1.5

Virus MC1061 3.3 1.5SV40-HB simian virus 40 JM103 3.6 1.6RSV-G respiratory tracts

Syncytial virus JM103 7.3 7.5HPV6B human papilloma JM103 10.9 3.1-URR virus 6b type MC1061 5.1 2.3HPV16 human papilloma-URR virus 16 type JM103 8.0 4.5JM103-M intestinal bacteria JM103 5.5 3.1

MC1061 5.4 2.8

C600 5.0 2.4

HB101 1.5 1.3

Embodiment 12 intestinal bacteria strengthen the increment sequence and have promoter activity

Application start detects carrier pKK232-8 plasmid, present inventors find that SV40 DNA Hind III B fragment also has stronger promoter function, the anti-paraxin of CAT genetic expression that can make its downstream is up to 700ug/mL, simultaneously prove that also HindIII K fragment also has very strong promoter function, can make the CAT genetic expression in its downstream, can anti-paraxin up to 900ug/ml.

Embodiment 13. in addition, in promoter detection carrier pKK232-8, recombinant plasmid has produced chlorampenicol resistant to the present inventor the JM103-M fragment cloning, can anti-paraxin up to 700ug/ml, this explanation JM103-M fragment has promoter function.

Embodiment 14. adopts and proves also that with quadrat method the enhancing increment sequence of HPV-6B, HPV16 and RSV-G also has the escherichia coli promoter function.

Embodiment 15. adopts and has proved that with the quadrat method present inventors above-mentioned enhancing increment sequence itself has promoter function simultaneously, but has the sequence of escherichia coli promoter function, and the increment of enhancing effect might not be arranged, and the results are shown in Table 2.

The active promoter activity VV-K of the promoter activity enhancer of table 2 pronucleus enhancer sample sequence or promoter enhancer++ SV40-HB++ PSV-G++ HPV6b-URR++ HPV16-URR++ M++ VV-7.5-+VV-HI-+VV-HN-+

Reinforcing effect and tandem promoter that embodiment 16. intestinal bacteria strengthen the increment sequence have nothing to do

JM-103 is inserted between the 7.5Kb promotor and CAT gene of pG (+) carrier, make and be arranged as: 5 ' p7.5-JM103-M-CAT 3 ', wherein distance is＞300bp between p7.5 and the M, the CAT expression of gene is very low as a result, otherwise, 5 '-JM103-M-p7.5-CAT 3 ', then genetic expression obviously strengthens, the enhanser effect remote series connection of two kinds of promotors no thanks to of this explanation M.

In addition, PRPL and upstream regulatory region X with enhanser effect thereof are placed the upstream of trp promotor, make its be arranged as 5 '-X--PRPL+trp-IFN-β gene-3 ', distance between wherein X-PRPL and trp further is 300bp, because PRPL has temperature effective in the lysogenic bacteria of clts857, the low efficient expression of cI gene in the time of 30 ℃, PRPL promotor efficient is extremely low, in the time of 42 ℃, cI genetic expression is obstructed, PRPL effectively works, and still, the result shows, the X-PRPL sequence does not have temperature effective, promptly X-PRPL-trp promoter expression beta-interferon has very strong reinforcing effect in the time of 37 ℃, and simple PRPL promoter expression beta-interferon, the expression amount in the time of 37 ℃ is very low.This explanation X-PRPL is to the enhancement of trp promotor the series connection effect of promotor but a kind of effect that strengthens increment no thanks to.

Implement the relation of 17. enhancing increment sequences and promotor type

The intestinal bacteria of above-mentioned animal virus are strengthened increment sequence insertion vaccinia virus promotor, and PL, trp promotor upstream detection they transcribe enhancement, the result shows that these intestinal bacteria strengthen not only the vaccinia virus promotor to be had in the sample sequence and strengthens the increment effect, to PL, the trp promotor has the increment of enhancing effect too.

Embodiment 18. intestinal bacteria strengthen increment sequence B EL does not have strict promotor specificity

Have two class promotors in intestinal bacteria at least, a class is σ 54 subunits that need RNA polymerase, and another kind of is to need σ 70 subunits.For inquiring into intestinal bacteria " enhanser " whether strict promotor specificity is arranged.Present inventors' synthetic the upstream regulatory region (having strengthened BEL) of 52bp gln Ap2 gene promoter (σ 54), the result proves that the BEL of 52bp is to trp (σ 70) and VV (σ?) the enhanser effect all arranged.

The enhancing increment sequence of synthetic intestinal bacteria glutamine synthetase gene upstream 51bp (Bacterial enhancer-like seqnence) (BEL).This enhanser is that active function is the strongest, most important one section in the complete sequence, and the synthetic base of 5 ' end is 42, comprises two protection bases and the HindIII site is set; The synthetic base of 3 ' end is 40, comprises two protection bases and the EcoRI site is set; Middle overlapping 15 bases, remaining mends flat with the extension of Partial K lenow enzyme.

The EcoRI-ClaI site that synthetic enhanser BEL is purified, carrier PATNF 153 is inserted with the EcoRI-HindIII end in the flat back of sex change, annealing, benefit, the characteristics of this recombinant plasmid are the upstreams that BEL places the trp promotor, tumour necrosis factor (TNF) gene is for detecting index, through restriction analysis and nucleotide sequencing, the confirmation recombinant plasmid is set up successfully.Do active the detection by TNF, find that its activity reaches 58.5MU/L recombinant plasmid PATNF153B, higher 5 times than maternal plant (PATNF153); Present inventors have been set up recombinant plasmid PAL-B again then, and its feature is that BEL places vaccinia virus 7.5K protein gene promoter upstream, and beta-galactosidase gene is for detecting gene.Mensuration to betagalactosidase activity shows that the specific activity PAL of PAL-3 is high 4 times.In addition, does the result show that also the BEL of 51bp is to trp (sigma70) promotor and vaccinia virus 7.5K gene promoter (sigma?) all have and strengthen the increment effect.

Embodiment 19. strengthens the increment sequence certain host specificity

Relatively M enhanser and HPV16-14U strengthen the increment sequence at JM103, MC1061, C600, in HB101, JM83 and the 7118 host bacterium to the enhancement of Lac genetic expression, the effect that found that HB101 is the poorest, and JM83 is the strongest, and certain host specificity has been described.

Embodiment 20.M sequence is to the reinforcing effect of transcribing of vaccinia virus promotor

Forward the M sequence placed the upstream of vaccinia virus promotor, no matter still oppositely all can make lac Z expression of gene strengthen 2.6-6 doubly.In order to prove that further the M sequence is to occur on the transcriptional level to lac Z expression of gene enhancement, the dna fragmentation of the part lac Z gene of present inventors' usefulness labelled with radioisotope is as probe, with Dot hybridization and Northern hybridizing method to carrying segmental lac ZmRNA assay and the comparison of cloning bacterium of M, the scanning result of DOT hybridization ARG shows, carry in the segmental clone of the M bacterium mRNA content than about 7 times of the segmental height of no M, similar to the betagalactosidase activity detected result, and the radioautograph result of Northern hybridization shows, the hybrid belt that carries the segmental clone of M bacterium lac ZmRNA formation also obviously increases than not carrying the segmental intensity of M.

This explanation M sequence occurs on the transcriptional level really to the enhancement of lac Z genetic expression, and the M sequence has the character that the intestinal bacteria enhanser is wanted sequence.

Embodiment 21. intestinal bacteria M sequences are to the reinforcing effect of transcribing of trp promotor

The M sequence is inserted into the upstream of intestinal bacteria trp promotor with positive and negative both direction, select for use people's α-Zhong Liuhuaisiyinzi (hTNF-α) as reporter gene simultaneously, make up the TNF expression plasmid (pATNFM (+) and pATNFM (-)) that has the M sequence, the influence of trp promoter transcription level has been studied the pronucleus enhancer function of M sequence by quantitative analysis M sequence.

The result shows, the specific activity that has the TNF that the expression plasmid of M sequence expresses is not with sequence person obviously to raise, wherein the TNF level that M sequence forward inserts and the reverse expression plasmid that inserts is expressed is about 4 times and 2 times that are not with M sequence person respectively, shows that the M sequence also has the effect of the enhanced of expression for the strong promoter trp promotor of intestinal bacteria itself.TNFcDNA with [α-32P] mark is that probe carries out Northern hybridization, relatively their transcriptional level can be found: the transcriptional level of the TNF mRNA of pATNFM (+) and pATNFM (-) is apparently higher than pATNF153, and prompting M sequence has the enhancement of transcribing for the trp promotor.

Embodiment 22.M strengthens the primary structure of increment sequence

Sequential analysis shows that the M fragment is intestinal bacteria (JM103) coding methionine(Met) (Methionine amio peptid) and 30s Ribosomal proteinS2 (PPSB) portion gene and upstream region of gene control region.The segmental complete sequence of M is seen accompanying drawing 6.

The research at the functional position of embodiment 23.M sequence

In order to determine the functional part of M sequence, present inventors have carried out the research of deletion mutant.From a series of mutant of M sequence forward clone 3 ' end, select 12 clones, from M sequence reverse cloning 3 ' a series of mutant of end, select 5 clones, deletion fragment is cloned into pAC detects carrier, measure the betagalactosidase activity of different deletion mutants, presentation of results forward deletion mutant 3 ' end disappearance 450bp is to not influence of enhanced activity, active decline lacks to the 640bp place to the 500bp place, and enhanced activity disappears.Inverse transition 3 ' end disappearance 100bp does not have influence to enhanced activity, lacks to the reduction of 250bp enhanced activity, and when lacking to 430bp, enhanced activity disappears.Illustrate that forward and inverse transition body result of study are identical substantially.

To studies show that of M sequence, reverse deletion mutant, the enhancement site is present in the 450bp sequence of forward clone 5 ' end upstream.

Embodiment 24. usefulness gel electrophoresises postpone the conjugated protein of experimental study M sequence

Whether each component of albumen and the M sequence extracted for preliminary understanding present inventors exist keying action, present inventors have carried out gel electrophoresis and have postponed experiment and competitive gel electrophoresis delay experiment, the result shows that ammonium sulfate fractionated each component has Degradation to dna probe, and at 0.65M NH ₄The part of Cl wash-out and dna probe in conjunction with after can produce three kinds of different Protein-DNA probes migration bands, postpone to experiment showed, in three binding substancess have a keying action the strongest that one is taken second place through competitive gel electrophoresis, one the most weak.In the presence of the 5ug competitive inhibitor, there are two combined belts to die down, and band completely dissolve when the 10ug competitive inhibitor exists is wherein arranged.RNA polymerase holoenzyme purifying thing can with the M fragment in conjunction with forming the band that mobility is different, host's conformity gene (IHF) can form very weak combined belt with the M fragment.

Embodiment 25. usefulness in-vitro transcription experimental study M sequences conjugated protein

Exist and M sequence bonded albumen in the intestinal bacteria JM103 tropina that utilization gel electrophoresis delay test and competitive gel electrophoresis delay test proof are extracted, present inventors utilize the pAC plasmid of M sequence forward clone's pASM (+) and no M sequence as template, in the presence of the conjugated protein and RNA polymerase that extracts, carried out the in-vitro transcription experiment.The in-vitro transcription experimental result shows, is that two re-reading systems of template all a band occurs near the place 3kbRNAMARK with pASM (+) and pAC respectively, and with pASM (+) be template to transcribe the band color darker, pAC system band color is more shallow.The in-vitro transcription experimental result shows that the M sequence of extraction is conjugated protein can not only to be combined with the M sequence with the RNA polymerase, and enhancement is transcribed in the performance that can also interact.

Embodiment 26. has establishment and the characteristics that intestinal bacteria strengthen the pBV320 serial carrier plasmid (pBV320, pBV321 and pBV322) of increment sequence X

PBV320 system escherichia coli high-level expression plasmid, be characterized in having the X sequence of 800bp length at 300bp place, trp promotor upstream, be lambda particles phage PRPL and upstream regulatory region thereof, there are multienzyme point of contacts such as Xbal, BamHI, Sal I, Pst I in trp promotor downstream, and the plasmid size is 4012bp.Difference is that the multienzyme point of contact is different to pBV320 with pBV321, and the multienzyme point of contact of pBV321 and pBV322 is identical, and difference is the EcoRI site disappearance at 290bp place, latter trp promotor upstream.The plasmid figure of pBV320 and pBV321 sees accompanying drawing 8,9.

Embodiment 27. has pBV324 serial carrier plasmid (pBV324, characteristics pBV325) that intestinal bacteria strengthen increment sequence M

The characteristics of pBV324 escherichia coli high-level expression carrier are to have the long intestinal bacteria of 908bp to strengthen increment sequence M fragment at PL promotor upstream end, in PL promotor downstream EcoRI are arranged, multienzyme point of contacts such as Smal I and BamH I and Pst I.

The characteristics of pBV325 escherichia coli high-level expression carrier are to have the long intestinal bacteria of 908bp to strengthen increment sequence M fragment at 290bp place, trp promotor upstream, in trp promotor downstream Xbal are arranged, multienzyme commonly used point of contacts such as Smal I and Pst I.The plasmid figure of pBV324 sees accompanying drawing 10.

Embodiment 28. utilizes intestinal bacteria to strengthen increment sequence carrier high-efficiency expressing human interferon-

Human beta interferon cDNA 5 ' holds the site into xba I, 3 ' hold the site into Bgl II, total length 504bp, carrier is pBV320, during establishment, earlier pBV320 is cut with Xba I and BamH I, then, through the T4DNA ligase enzyme, with the two connection, transformed into escherichia coli JM103, cut through enzyme, in situ hybridization, Sourthen identifies errorless, obtains pBV320-β and efficiently expresses bacterial classification.The level that pBV320-β expresses Interferon, rabbit is higher 7.3 times than pBV220-β.

Embodiment 29. utilizes intestinal bacteria to strengthen increment sequence carrier high-efficiency expressing human IFN-

Carrier adopts the pBV322 that has the intestinal bacteria enhancer sequence, with Xba I the pBV322 enzyme is cut, IFN-cDNA takes from pBV220 γ, and IFN-cDNA two ends are EcoR I site, carrier, fragment is mended flat through KLenow, the T4DNA ligase enzyme connects then, transforms JM103, cuts through enzyme, molecular hybridization is identified correct, and the expression level pBV220 γ that obtains pBV320 γ efficient expression strain .pBV320 γ is high 7.3 times.

It is to efficiently express human tumor necrosis factor-a to take off the TNFcDNA that takes up the beginning codon and removed signal peptide coding region from plasmid pTNF8 with restriction enzyme Xbal and PstI intestinal bacteria that embodiment 30. utilizes the novel carriers that has pronucleus enhancer sample sequence X and M, be inserted into the corresponding site of pbV320, make 5 of TNFcDNA ' end directly place promotor trp downstream, identify through hybridization, restriction analysis, obtain recombinant plasmid pBV320/TNF-α.Institute obtains and the results are shown in Table 3.

The different TNF plasmid vectors of table 3 have 135.20+16.92pBV220/TNF PRPL not have 63.42+24.15pBV320 trp at comparison plasmid promotor pronucleus enhancer sequence X TNF activity (MU/l) * of expression in escherichia coli level pBV320/TNF trp has 0.00pBV320 PRPL not have 0.00

Fire TNF activity is the mean number of 5 experiments, and the host bacterium is JM103.

From the pBV320/TNF plasmid, downcut the fragment that about 1.0kb contains trp promotor and tnf gene with restriction enzyme EcoR I and Pst I in addition, be inserted into the corresponding restriction enzyme site of pAT 153 plasmids, obtain TNF expression vector pATNF 153.From PGM (+) plasmid, downcut the M fragment of about 0.9kb with restriction endonuclease Hind III, the Hind III enzyme point of contact (making the M fragment be positioned at trp promotor upstream) that is inserted into pATNF153 obtains the TNF expression vector pATNFM (+) of positive and negative two direction of insertion, pATNFM (-).Expression level is seen 4 tables.

The different TNF plasmid vectors of table 4 are in the comparison plasmid promoter enhancer TNF of expression in escherichia coli level activity, (u/L) pATNFM trp has, (M+) 1.8+0.11 * 10pATNFM trp has, (M-) 0.91 ± 0.13 * 10pATNF trp does not have 0.45 ± 0.16 * 10pBV220/TNF PRPL and does not have 0.63 ± 0.14 * 10n=5 Host Strains JM103; The M+ forward inserts the M fragment, and M-oppositely inserts M fragment p＜0.05

Embodiment 31. utilizes intestinal bacteria and strengthens increment sequence carrier high-efficiency expression human.

Human cDNA two ends are EcoR I site, and length is 565bp, and 5 ' end EcoR I is ATG behind the point of contact, then is the nucleotide sequence of coding 166 amino acids.During establishment, earlier pBV322 is cut with Xba I and EcoR I, the Klenow enzyme is mended flat; Simultaneously, two ends are that the Interferon, rabbit fragment of EcoR I is cut with EcoR I, also mend flatly with the Klenow enzyme, and pBVXalb is set up in the two connection, through enzyme cut identify errorless, transformed into escherichia coli JM103.

The comparison of pBVXalb and pBV867 expression level: under similarity condition, compared novel pronucleus enhancer expression vector pBVXalb and original production bacterial classification pBV867 in the level of shaking bottle expression Interferon, rabbit.The results are shown in Table 5.

Table 5 human expression level (MU/L) pBV867 pBVXalb0.41 30.40.53 26.20.48 52.40.48 52.40.68 42.60.68 45.00.58 45.10.68 30.40.55 40.6

Plasmid pBVXalb passed for 100 generations under laboratory condition, the level of results expression Interferon, rabbit is not seen obvious change.Passage number Interferon, rabbit (MU/L) 1 13,360 12,370 12,380 12,990 123,100 129 that tire

Embodiment 32. utilizes intestinal bacteria to strengthen increment sequence carrier high-efficiency expressing human α 2b type Interferon, rabbit

To contain the Xba I-Pst I site that the EcoR I-Pst I fragment of the full gene of IFN-α 2b is inserted escherichia coli efficient expression vector pBV321, obtain the efficient expression vector pBV889 of IFN-α 2b.The host bacterium is JN103.Insert fragment and identify and after making nucleic acid molecular hybridization proves that size and direction are errorless,, and express at 37 ℃ with pBV889 transformed into escherichia coli JM103 strain through restriction enzyme digestion.Having the average expression level that protokaryon strengthens carrier is 18000mu/L, and the average expression level that does not have an enhanser carrier is 200mu/L.After passing for 100 generations, the interferon expression level is constant.

Embodiment 33. utilizes intestinal bacteria to strengthen increment sequence carrier high-efficiency and expresses efficiently expressing of humanIFN-2a

Present inventors downcut the people α 2a type interferon gene of 865bp from the pBV888 plasmid with EcoR I and Pst I; Simultaneously, cut pBV320, both are connected with dna ligase with Xbal and Pst I, and then flat with Klenow enzyme benefit, connect again, enzyme is cut evaluation, and transformed into escherichia coli JM103 is built into pBV918.Present inventors are containing on 100ug/ml penbritin 1.3% agar plate 60 generations of continuous passage with the pBV918 bacterial classification, and 10 generations of every biography are extracted plasmid after getting actication of culture, identify with the inscribe enzymolysis, and measure biologic activity on WISH cell, the results are shown in Table 6.Restriction endonuclease assay certificate plasmid is not lost after 60 generations, and still can keep efficiently expressing human interferon-alpha 2a, and average expression amount is 2Kmu/l.

The different horizontal algebraically interferon activity of algebraically bacterial classification interferon expression (Mega unit per liter) of table 6 pBV918 230860 generations 2651 of 284150 generations, 221440 generations, 218330 generations, 209420 generations, 212410 generations, 1 generation

People rIFN-α 2a behind the monoclonal antibody purifying uses Tris/HCl, and pH 7.5 balances are placed half a year at 4 ℃, checks its activity in every month, and titre is stable in half a year.Institute obtains and the results are shown in Table 7.

Table 7 people rIFN-α 2a is to the Interferon, rabbit titres 4 ℃ of following storage periods of stability (moon) (MIU/ml) of temperature

0 2730

1 2620

2 2140

3 2370

4 2010

5 2520

6 2290

Embodiment 34. present inventors utilize intestinal bacteria M and X " enhanser " sequence to reconstruct two kinds of efficient expression vectors, have successfully improved 6 kinds of expression of gene amounts.Institute obtains and the results are shown in Table 8.

Table 8 Escherichia coli strengthen the increment sequence and efficiently express various active peptide carrier foreign gene promoter enhancer resistance activity pBV220 IFN-α 2a PRPL-Amp 1.0pBV320 IFN-α 2a trp * Amp 4.6pBV320 IFN-α 2b trp-Tet 1.0pBV320 IFN-α 2b trp * Tet 17.3pBV220 IFN-α Ib/86D PRPL-Amp 1.0pBV321 IFN-α Ib/86D trp * Amp 3.0pBV320 IFN-α Ib/86c trp-Tet 1.0pBV321 IFN-α Ib/86c trp * Tet 4.6pBV220 IFN-γ PRPL-Amp 1.0pBV321 IFN-γ trp * Amp 7.3pBV320 TNF-α trp-Tet 1.0pBV324 TNF-α trp M+ Tet 4.0pBV324 TNF-α trp M-Tet 2.0

Reference (1) Banerji, J, et al., Cell, 27 (1981), 299. (2) Benoist, C.et al., Nature, 290 (1981), 304. (3) Harley, C.B.et al., NAR, 15 (1987), 2343. (4) Nishi, T.et al., Gene, 44 (1986), 29

(5)Buch，M.et?al.，Nature，320(1986)，374

(6)Garciarrabio.A.A.et?al.，Gene，54(1987)，275.

(7)Huber，H.E.et?al.，PNAS，82(1985)，3776

(8)Kahmann，R.et?al.，Cell，41(1985)，771.

(9)Katta，M.et?al.，DNA?and?Cell?Biol.，1990，9∶129.

Claims (6)

1, intestinal bacteria are originated, and the dna fragmentation with pronucleus enhancer sample function is characterized in that, said dna fragmentation has nucleotide sequence as shown in Figure 6.

2, SV40 viral source, the dna fragmentation with pronucleus enhancer sample function is characterized in that, said dna fragmentation has nucleotide sequence as shown in Figure 1.

3, vaccinia virus is originated, and the dna fragmentation with pronucleus enhancer sample function is characterized in that, said dna fragmentation has nucleotide sequence as shown in Figure 2.

4, RSV viral source, the dna fragmentation with pronucleus enhancer sample function is characterized in that, said dna fragmentation has nucleotide sequence as shown in Figure 3.

5, HPV16 viral source, the dna fragmentation with pronucleus enhancer sample function is characterized in that, said dna fragmentation has nucleotide sequence as shown in Figure 5.

6, the application of the dna fragmentation of claim 1 in making up recombinant expression vector.

Images