CN1673377A - Method for recombining human PSP94 protein secretory expression in colibacillus - Google Patents

Abstract

The present invention relates to method of secreting and expressing recombinant human PSP94 protein in colibaccillus, and belongs to the field of the expression of bioactive human PSP94 protein. The present invention includes extracting total prostate tissue RNA, obtaining Chinese PSP94 cDNA sequence via RT-PCR process, inserting it into cloned vector pUC19, determining sequence after coarse PCR screening; connecting double restricted PSP94 protein coding gene, signal peptide and pBV220 plasmid to constitute recombinant plasmid pBV-PSP94; transforming colibaccillus strain with the positive recombinant plasmid pBV-PSP94 and protein inducing expression at optimal temperature 40 deg.c for optimal time of 5 hr. The present invention has the beneficial effect of mutating the signal peptide of secreting expression vector and inducing restriction site to the signal peptide superiorly by means of molecular biological technology.

Description

The method of recombining human PSP 94 protein secreting, expressing in intestinal bacteria

Technical field

The present invention relates to a kind of method that adopts the intestinal bacteria secreting, expressing that bioactive human PSP 94 protein is arranged.

Background technology

Prostate cancer is the representative disease of elderly men.The sickness rate that played U.S.'s prostate cancer in 1996 has occupied first of male sex's malignant tumour, occupies second of cancer mortality and mortality ratio is only second to lung cancer.Because prostate position is hidden, cancer mostly occurs in the surrounding zone away from urethra, and modern detecting blood-serum P SA detects the needs that are difficult to satisfy early diagnosis, makes a lot of patients lose the chance of treatment.

So far, China's clinical prostate cancer is still and could diagnoses late period.Rose in 1999, this seminar place " prostatosis study on prevention " center " is under the assistance of Sino-Japan inter-governmental specific technique collaborative project; use blood-serum P SA to name surplus the Changchun 16500 more than 50 years old the male sex carried out the generaI investigation of prostate cancer group, the discovery rate of prostate cancer surpasses 1.7%.Wherein, the middle and advanced stage case accounts for 42%, accounts for 18.8% with the bone transferrer.We think that PSA has tangible specificity to prostate cancer diagnosis among the suspicious crowd of PSA＞10.0ng/ml.For example, the prostate cancer diagnosis rate surpasses 95% among the suspicious crowd of PSA content＞40.0ng/ml, and diagnosis surpasses 75% among the crowd of PSA＞20.0ng/ml.Yet, at PSA content 〉=4.0ng/ml-10.0ng/ml, suspicious crowd biopsy under ultrasonic guidance of so-called " grey area ", the diagnosis of prostate cancer is only about in the of 15%.Therefore, for improving the prostate cancer diagnosis efficient of PSA gray area, be badly in need of exploring new tumor markers.

In recent years the domestic and international research result shows, prostate epithelial cell synthesis secretion a kind of contained 94 amino acid whose albumen, and promptly PSP94 is having a good application prospect aspect treatment and the monitoring prostate cancer progress.Because of PSP94 mainly is present in the prostatic fluid, natural PSP94 is difficult to a large amount of the acquisition.At present the recombination fusion protein form is adopted in the expression of PSP94 more both at home and abroad, though this amalgamation and expression has advantages such as expression efficiency is higher, proteolytic degradation is less, but need enzyme to cut or chemical cracking removal fusion rotein after expressing, and the albumen that produces exists with the inclusion body form of non-activity, the process that needs external sex change repeatability has increased the workload in product downstream greatly.Because PSP94 is most Gelucystine albumen, external renaturation is difficult to form correct disulfide linkage, the proteic almost lifeless matter of gained activity.

Existing protein excretion expression technology is to connect multiple clone site in the DNA downstream of vector encoded signal peptide sequence.After being connected into goal gene, the DNA of multiple clone site also translates into amino acid, thereby might make the secretory protein of expression lose biological activity, might cause as medicine the immune response of human body to produce various side effects.

Summary of the invention

To express aftertreatment loaded down with trivial details in order to overcome existing P SP94, and workload is big, and the deficiency that the biologically active prod yield is low the invention provides the method for a kind of recombining human PSP 94 protein secreting, expressing in intestinal bacteria.By transgenation to signal peptide, restriction enzyme site is designed into signal peptide inside, remove the unnecessary amino acid problem that multiple clone site causes.The recombinant protein direct secretion has not only been avoided the degraded of the interior various proteolytic enzyme of thalline to recombinant protein to the less periplasmic space of foreign protein, has increased expression level, is easy to purifying.And albumen can form disulfide linkage under the natural situation under the oxidized form environment of periplasmic space, directly obtains having the protein of natural structure, and cytologic experiment has proved its biologic activity.Adopt temperature-regulated expression, avoided using the cost of inductor.

The technical solution used in the present invention is:

One, the clonal expansion of PSP94 cDNA

Extract the total RNA of prostata tissue, angle by the RT-PCR method and get Chinese PSP94cDNA sequence, insert among the cloning vector pUC19, carry out sequencing after the PCR scalping;

Two, the structure of recombinant secretor type expression vector

PSP94 protein coding gene behind the double digestion, signal peptide and pBV220 plasmid are connected construction recombination plasmid pBV-PSP94;

Three, reorganization PSP94 protein expression

Positive recombinant plasmid pBV-PSP94 is the transformed into escherichia coli bacterial strain respectively, as JM109, HB101, LB21 bacterial strain, preferred JM109 expression effect is best, so PSP94 albumen is chosen JM109 and is expressed as the host bacterium, through groping protein induced expression optimum temperuture is 40 ℃, and optimum expression time is 5 hours.

The step 2 signal peptide is taked the natural diphtheria toxin signal peptide of modified mistake.

The natural diphtheria toxin signal peptide of the modified mistake of step 2 is:

ES1 is that the signal peptide that contains EcoR I restriction enzyme site is had a mind to strand primer:

5’-CGGAATTCGTGAGCAGAAAACT-3’

ES2：5’-GATGC?ATG?CGC?TGA?AGG?TGG?GGC?CCC?TAT?CCCCAG?TAG?CGC?CCC?TAT?TAA?GAT?TGA?CGC?AAA?CAG?TTT?TCTGCT?CAC?GAATTCCG-3’

We utilize the pBV220 plasmid as expression vector, and it contains the strong promoter P that is subjected to temperature adjusting _RP _LIntroduced the diphtheria toxin signal peptide sequence of transforming.Utilize the merger of amino acid code word, promptly utilize the corresponding a kind of amino acid whose character of a plurality of codons of genes encoding, the gene of the signal peptide of sudden change diphtheria toxin under the prerequisite that does not change aminoacid sequence, restriction endonuclease sites SphI is incorporated in the signal peptide sequence, is convenient to the connection of PSP94 gene.Utilize the RT-PCR technology PSP94 gene of from human prostate tissue, cloning people.Adopt round pcr to introduce the SphI site at the N of PSP94 protein coding gene end.The C end is introduced the restriction endonuclease sites that connects and has been designed two terminator codons, can guarantee the correct termination of protein translation.Cut through enzyme, be connected into the carrier that contains the diphtheria toxin signal peptide sequence after the transformation.Screening has the cloned plasmids that inserts gene.The bacterial strain of optimization expression and expression condition.Utilize the character of holding back of semi-permeable membranes, separation and purification PSP94.Adopt the SDS-PAGE electrophoretic technique to check its purity, cytologic experiment is verified its biologic activity.

The invention has the beneficial effects as follows, pass through Protocols in Molecular Biology, the signal peptide of sudden change secretion expression carrier, on signal peptide, introduce restriction enzyme site, overcome traditional secretion expression carrier and before expressed proteins, added unnecessary amino acid whose problem, obtain primary structure and the identical product of human PSP 94 native protein, made the PSP94 of expression in human body, not have immunological rejection, might become the medicine of new anti-prostate cancer.The PSP94 secretive expression vector that reorganization obtains can make the recombinant protein of expression be secreted in the colibacillus periplasm space, avoided the Degradation of thalline endoproteinase, the atomic albumen of thalline of periplasmic space intensive amount own makes the separation and purification of recombinant protein be more prone to again, and the oxidized form environment in the periplasmic space can also be simulated eukaryotic endoplasmic reticulum, form the disulfide linkage in the natural PS P94 albumen, make the albumen space structure more near native state.This phraseology has been avoided traditional inclusion body technology complicated renaturation purge process behind the expressing protein in thalline, and recombinant protein also has higher biologic activity.On the basis that obtains the secretor type recombinant protein, again by active checking proof, recombinant protein has very obvious suppression cell proliferation, promote apoptosis and make the activity of the optimum conversion of cancer cells

Description of drawings

Fig. 1, be depicted as the agarose gel electrophoresis result of RT-PCR product, about 480bp, locate to see a tangible band,

Fig. 2, recombinant plasmid PCR checking is figure as a result, has located an obvious DNA band about 280bp, and is consistent with PSP94 protein coding gene length.

Fig. 3, recombinant plasmid dna sequencer map, underscore partly are PSP94 cDNA sequence, and be identical with expected results.

Fig. 4, structure recombinant secretor type expression plasmid pBV-PSP94 schema.

The PCR scalping rear electrophoresis of Fig. 5, recombinant plasmid pBV-PSP220 is figure as a result.

The sequencing result figure of Fig. 6, recombinant plasmid pBV-PSP94.

Fig. 7, the proteic SDS-PAGE of recombinant secretor type PSP94 and Western blot analyze.

PC-3 cell inhibiting rate behind Fig. 8, the various dose PSP94 processing 48h.

Fig. 9, PSP94 handle different time for PC-3 cell inhibiting rate.

Figure 10, a, b part, the morphological change of PC-3 cell behind the reorganization PSP94 albumen processing 48h.

Figure 11, PSP94 handle back PC-3 cell DNA content and change histogram.

The physical map of Figure 12, PBV220.

Embodiment

Material, bacterial strain, cell strain and reagent

The normal prostate tissue prostate cancer tissue of choosing provide by clinical medicine institute of Jilin University Urology Surgery.

The various tool enzyme comprises that reversed transcriptive enzyme, restriction enzyme, ligase enzyme, TaqDNA polysaccharase are TaKaRa company product.

Dna gel reclaims test kit available from TaKaRa company, and the protein quantification test kit is a Bio-Rad company product.

Bacterial strain E.coli JM109 and plasmid pUC19, pBV220 are by the preservation of going down to posterity of this laboratory.E.coli JM109 and plasmid pUC19 are the commodity that can buy, about complete sequence and physical map are provided in the pBV220 present embodiment.

The PC-3 cell strain is provided by Urology Surgery institute of former Beijing Medical University, can buy.

After various primers design voluntarily, synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, comprise respectively: primer CP1 and the CP2 of RT-PCR amplification PSP94 cDNA; Ripe PSP94 protein gene primer EP1 and EP2 and signal peptide sequence ES1 and ES2.

Other conventional chemical reagent is import or homemade analytical pure product.

Embodiment:

One, the clonal expansion of PSP94 cDNA

Utilizing the guanidinium isothiocyanate single stage method to extract the total RNA of people's normal prostate tissue, under primer Oligo (dT) 15 guiding, through the synthetic cDNA of AMV reversed transcriptive enzyme effect, is primer PCR amplification PSP94 cDNA with CP1, CP2 again.

CP1(5’TTACTGATAGGCATGGCTAC-3’)

CP2(5’-TGCTTATCACAATGAATGTTCTCCTGGGG-3’)

The amplified production gel reclaims the back and is connected with the pUC19 plasmid of cutting through the SmaI enzyme, makes up cloned plasmids pUC-PSP94.Connect product transformed competence colibacillus and E.coli JM109 bacterium, be laid on the LB agar plate that contains penbritin, IPTG and X-Gal, after 37 ℃ of inversion overnight incubation, select white colony to be inoculated in the LB liquid nutrient medium that contains the ammonia benzyl, after 37 ℃ of shaking culture are spent the night, extract plasmid in a small amount with alkaline lysis, with EP1 and EP2 is that primer amplification PSP94 protein gene fragment is carried out scalping to positive recombinant plasmid, afterwards the recombinant plasmid of selecting is served sea living worker ox thing engineering Services Co., Ltd and is carried out sequencing.

EP1(5’-GCGCATGCATCATGCTATTTCATACCTAAT-3’)

EP2(5’-CGGGATCCTTATCAGATTATCCATTCACT-3’)

The pUC-PSP94 plasmid that order-checking is proved correct reorganization is a primer amplification PSP94 protein gene with EP1 and EP2, and behind the BamHI/SphI double digestion, gel reclaims standby.

Figure 1 shows that the agarose gel electrophoresis result of RT-PCR product, about 480bp, locate to see a tangible band, consistent with PSP94 cDNA sequence length.

Recombinant plasmid PCR checking the results are shown in Figure 2, has located an obvious DNA band about 280bp, and is consistent with PSP94 protein coding gene length.The recombinant plasmid dna sequencing result is seen Fig. 3, and underscore partly is PSP94 cDNA sequence, and is identical with expected results.Illustrating that we clone has obtained correct PSP94 cDNA sequence.

Two, the structure of recombinant secretor type expression vector

A.) design of signal peptide sequence

Natural diphtheria toxin signal peptide contains 25 amino acid, and the dna encoding sequence is:

GTG?AGC?AGA?AAA?CTG?TTT?GCG?TCA?ATC?TTA?ATA?GGG?GCGCTA?CTG?GGG?ATA?GGG?GCC?CCA?CCT?TCA

-1 ,-2 and-3 of natural diphtheria toxin signal peptide aminoacid sequence, be that aminoacid sequence Ala-His-Ala constitutes the recognition site of signal peptidase (the coding base sequence is GCCCATGCA, shown in the square frame), after diphtheria toxin is secreted into outside the born of the same parents, signal peptidase from then on site cuts signal peptide sequence, only stays mature protein.

According to the native sequences of diphtheria toxin, designed the signal peptide sequence ES1 and the ES2 of following secretion PSP94 recombinant protein.

ES1：5’-CGGAATTCGTGAGCAGAAAACT-3’

ES2：5’GA TGC?ATG?CGC?TGA?AGG?TGG?GGC?CCC?TAT?CCC CAG?TAG?CGC?CCC?TAT?TAA?GAT?TGA?CGC?AAA?CAG?TTT?TCT GCT?CAC?GAATTCCG-3’

In the above-mentioned signal peptide primer, ES1 is that the signal peptide that contains EcoR I restriction enzyme site is had a mind to strand primer, underscore partly is a diphtheria toxin signal peptide complete sequence among the ES2, with the only difference of urao basic sequence be under the prerequisite that does not influence coded amino acid (L-Ala), the codon of signal peptide-3 amino acids is become GCG by GCC.Simultaneously, Sph I restriction enzyme site (GCATGC) has also been introduced in this change, just the site that signal peptide is connected with the PSP94 encoding gene is designed into signal peptide sequence inside, make that protein excretion signal peptide behind the periplasmic space is cut fully, the unnecessary amino acid problem that the joint causes when having avoided making up recombinant protein.

ES1 and ES2 are material with dNTP under the effect of Klenow archaeal dna polymerase, and room temperature is extended, and product is behind the EcoRI/SphI double digestion, and agarose gel electrophoresis reclaims standby.

B.) structure of recombining human PSP 94 secretive expression vector

Plasmid pBV220 is behind the BamHI/EcoRI double digestion, and PSP94 protein gene product and signal peptide with above-mentioned double digestion connect under the effect of T4 dna ligase, makes up recombinant secretor type expression plasmid pBV-PSP94.Make up flow process as shown in Figure 4.PBV220 plasmid sequence total length (3666bp):

1? aattcccgg g?gatccgtcga?cctgcagcca?agcttctgtt?ttggcggatg?agagaagatt

61?ttcagcctga?tacagattaa?atcagaacgc?agaagcggtc?tgataaaaca?gaatttgcct

121?cccggcagta?gcgcggtggt?cccacctgac?cccatgccga?actcagaagt?gaaacgccgt

181?agcgccgatg?gtagtgtggg?gtctccccat?gcgagagtag?ccaactgcca?ggcatcaaat

241?aaaacgaaag?gctcagtcga?aagactgggc?ctttcgtttt?atctgttgtt?tgtcggtgaa

301??cgctctcctg?agtaggacaa?atccgccggg?agcggatttg?aacgttgcga?agcaacggcc

361??cggagggtgg?cgggcaggac?gcccgccata?aactgccagg?catcaaatta?agcagaaggc

421??catcctgacg?gatggccttt?ttgcgtttct?acaaactctt?tgtttatttt?tctaaataca

481??ttcaaatatg?tatccgctca?tgagacaata?accctgataa?atgcttcaat?aatattgaaa

541??aaggaagagt?atgagtattc?aacatttccg?tgtcgccctt?attccctttt?ttgcggcatt

601??ttgccttcct?gtttttgctc?acccagaaac?gctggtgaaa?gtaaaagatg?ctgaagatca

661??gttgggtgca?cgagtgggtt?acatcgaact?ggatctcaac?agcggtaaga?tccttgagag

721??ttttcgcccc?gaagaacgtt?ttccaatgat?gagcactttt?aaagttctgc?tatgtggcgc

781??ggtattatcc?cgtgttgacg?ccgggcaaga?gcaactcggt?cgccgcatac?actattctca

841??gaatgacttg?gttgagtact?caccagtcac?agaaaagcat?cttacggatg?gcatgacagt

901??aagagaatta?tgcagtgctg?ccataaccat?gagtgataac?actgcggcca?acttacttct

961??gacaacgatc?gggaggaccg?aaggagctaa?ccgctttttt?gcacaacatg?ggggatcatg

1021??taactcgcct?tgatcgttgg?gaaccggatc?tgaatgaagc?cataccaaac?gacgagcgtg

1081??acaccacgat?gcctgtagca?atggcaacaa?cgttgcgcaa?actattaact?ggcgaactac

1141??ttactctagc?ttcccggcaa?caattaatag?actggatgga?ggcggataaa?gttgcaggac

1201??cacttctgcg?ctcggccctt?ccggctggct?ggtttattgc?tgataaatct?ggagccggtg

1261??agcgtgggtc?tcgcggtatc?attgcagcac?tggggccaga?tggtaagccc?tcccgtatcg

1321??tagttatcta?cacgacgggg?agtcaggcaa?ctatggatga?acgaaataga?cagatcgctg

1381??agataggtgc?ctcactgatt?aagcattggt?aactgtcaga?ccaagtttac?tcatatatac

1441??tttagattga?tttaaaactt?catttttaat?ttaaaaggat?ctaggtgaag?atcctttttg

1501??ataatctcat?gaccaaaatc?ccttaacgtg?agttttcgtt?ccactgagcg?tcagaccccg

1561??tagaaaagat?caaaggatct?tcttgagatc?ctttttttct?gcgcgtaatc?tgctgcttgc

1621??aaacaaaaaa?accaccgcta?ccagcggtgg?tttgtttgcc?ggatcaagag?ctaccaactc

1681??tttttccgaa?ggtaactggc?ttcagcagag?cgcagatacc?aaatactgtc?cttctagtgt

1741??agccgtagtt?aggccaccac?ttcaagaact?ctgtagcacc?gcctacatac?ctcgctctgc

1801??taatcctgtt?accagtggct?gctgccagtg?gcgataagtc?gtgtcttacc?gggttggact

1861??caagacgata?gttaccggat?aaggcgcagc?ggtcgggctg?aacggggggt?tcgtgcacac

1921??agcccagctt?ggagcgaacg?acctacaccg?aactgagata?cctacagcgt?gagcattgag

1981??aaagcgccac?gcttcccgaa?gggagaaagg?cggacaggta?tccggtaagc?ggcagggtcg

2041??gaacaggaga?gcgcacgagg?gagcttccag?ggggaaacgc?ctggtatctt?tatagtcctg

2101??tcgggtttcg?ccacctctga?cttgagcgtc?gatttttgtg?atgctcgtca?ggggggcgga

2161??gcctatggaa?aaacgccagc?aacgcggcct?ttttacggtt?cctggccttt?tgctggcctt

2221??ttgctcacat?gttctttcct?gcgttatccc?ctgattctgt?ggataaccgt?attaccgcct

2281??ttgagtgagc?tgataccgct?cgccgcagcc?gaacgaccga?gcgcagcgag?tcagtgagcg

2341??aggaagcgga?agagcgccct?tatctttccc?tttatttttg?ctgcggtaag?tcgcataaaa

2401??accattcttc?ataattcaat?ccatttacta?tgttatgttc?tgaggggagt?gaaaattccc

2461??ctaattcgat?gaagattctt?gctcaattgt?tatcagctat?gcgccgacca?gaacaccttg

2521??ccgatcagcc?aaacgtctct?tcaggccact?gactagcgat?aactttcccc?acaacggaac

2581??aactctcatt?gcatgggatc?attgggtact?gtgggtttag?tggttgtaaa?aacacctgac

2641??cgctatccct?gatcagtttc?ttgaaggtaa?actcatcacc?cccaagtctg?gctatgcaga

2701??aatcacctgg?ctcaacagcc?tgctcagggt?caacgagaat?taacattccg?tcaggaaagc

2761??ttggcttgga?gcctgttggt?gcggtcatgg?aattaccttc?aacctcaagc?cagaatgcag

2821??aatcactggc?ttttttggtt?gtgcttaccc?atctctccgc?atcacctttg?gtaaaggttc

2881??taagcttagg?tgagaacatc?cctgcctgaa?catgagaaaa?aacagggtac?tcatactcac

2941??ttctaagtga?cggctgcata?ctaaccgctt?catacatctc?gtagatttct?ctggcgattg

3001?aagggctaaa?ttcttcaacg?ctaactttga?gaatttttgc?aagcaatgcg?gcgttataag

3061?catttaatgc?attgatgcca?ttaaataaag?caccaacgcc?tgactgcccc?atccccatct

3121?tgtctgcgac?agattcctgg?gataagccaa?gttcattttt?ctttttttca?taaattgctt

3181?taaggcgacg?tgcgtcctca?agctgctctt?gtgttaatgg?tttctttttt?gtgctcatac

3241?gttaaatcta?tcaccgcaag?ggataaatat?ctaacaccgt?gcgtgttgac?tattttacct

3301?ctggcggtga?taatggttgc?atgtactaag?gaggttgtat?ggaacaacgc?ataaccctga

3361?aagattatgc?aatgcgcttt?gggcaaacca?agacagctaa?aagatctctc?acctaccaaa

3421?caatgccccc?ctgcaaaaaa?taaattcata?taaaaaacat?acagataacc?atctgcggtg

3481?ataaattatc?tctggcggtg?ttgacataaa?taccactggc?ggtgatactg?agcacatcag

3541?caggacgcac?tgaccaccat?gaaggtgacg?ctcttaaaaa?ttaagccctg?aagaagggca

3601?gcattcaaag?cagaaggctt?tggggtgtgt?gatacgaaac?gaagcattgg?ttaaaaatta

3661?aggagg

Signal peptide that inserts and PSP94 sequence (between the 5th and the 10th, be equivalent to remove 4 bases, insert 363 bases again, insert the long 4025bp of being of signal peptide and PSP94 cDNA recombinant plasmid afterwards):

aattc?GTG?AGC?AGA?AAA?CTG?TTT?GCG?TCA?ATC?TTA?ATA?GGG?GCG?CTA?CTG

GGG????ATA?GGG?GCC?CCA?CCT?TCA?GCG?CAT?GCA?TCATGCTATT?TCATACCTAA

TGAGGGAGTT?CCAGGAGATT?CAACCAGGAA?ATGCATGGAT?CTCAAAGGAA?ACAAACACCC

AATAAACTCG?GAGTGGCAGA?CTGACAACTG?TGAGACATGC?ACTTGCTACG?AAACAGAAAT

TTCATGTTGC?ACCCTTGTTT?CTACACCTGT?GGGTTATGAC?AAAGACAACT?GCCAAAGAAT

CTTCAAGAAG?GAGGACTGCA?AGTATATCGT?GGTGGAGAAG?AAGGACCCAA?AAAAGACCTG

TTCTGTCAGT?GAATGGATAA?TCTGATAA? ggatcc

Connect product transformed competence colibacillus E.coli JM109 bacterium, be laid on the LB agar plate that contains penbritin, after 31 ℃ of inversion incubated overnight, choose single bacterium colony, be inoculated in the LB liquid nutrient medium that contains penbritin, 31 ℃ of shaking culture are spent the night.In a small amount extract plasmid with alkaline lysis next day, and with the positive recombinant chou of BamHI/EcoRI double digestion scalping, electrophoresis result is seen Fig. 5, located an obvious DNA band about 380bp, and size is consistent with the length sum of PSP94 protein coding gene and signal peptide sequence.The scalping thing is served sea living worker's biotechnology Services Co., Ltd and is checked order.Sequencing result is seen Fig. 6,-3 coding of visible signal peptide sequence are GCG, no any unnecessary base between synchronous signal peptide sequence and the PSP94 albumen coded sequence, after signal peptidase cuts signal peptide sequence, can not retain any unnecessary amino acid at the PSP94 albumen n end, two terminator codons after the PSP94 protein sequence can guarantee effective termination of protein translation, and mode of connection is consistent with our design.

Three, reorganization PSP94 protein expression

A.) express

The JM109 bacterial classification that contains the pBV220 plasmid of positive recombinant plasmid pBV-PSP94 and sky is inoculated in 100ml respectively and contains in the LB liquid nutrient medium of ammonia benzyl, and 31 ℃ of shaking culture treat that bacterium liquid OD600 reaches at 0.6 o'clock, and temperature is transferred to 40 ℃, continues to cultivate 5h.

B.) purifying and evaluation

Above-mentioned bacterium liquid is at 12000rpm, 4 ℃ of centrifugal 10 minutes collection thalline, supernatant discarded, precipitation is suspended from the lysozyme soln of new preparation, ice bath 10 minutes, 10,000rpm, 4 ℃ of centrifugal 10 minutes collection supernatants, be the periplasmic space part, periplasmic space liquid carries out purifying by dialysis (molecular weight is 7000) and semi-permeable membranes centrifuging (molecular weight cut-off is 30KD) successively, the protein SDS-PAGE electrophoresis result is seen Fig. 7, the bacterial strain periplasmic space liquid that contains pBV-PSP94 has been located an obvious protein band about 11kD, consistent with the PSP94 molecular weight of albumen, on electrophorogram, almost show as single band behind the purifying, density analysis shows that this band behind the purifying can account for 93% of total protein behind the purifying, and almost can't see band at the 11kD place after containing the bacterium periplasmic space product purification of pBV220 empty plasmid, and Western blotting result shows that gained albumen is PSP94, the expression of recombinant proteins amount can reach 100mg/L.

Experimental example the present invention PSP94 protein-active analysis of recombinating

Recombinant protein after adopting Bio-Rad Protein Assay to purifying carries out quantitatively with the RPMI-1640 nutrient solution PSP94 furnishing concentration being respectively (1000,500,100,50,10,5,0) * 10 ^-4M, filtration sterilization.

(1) mtt assay is measured prostate cancer PC-3 cell concn is transferred to 2 * 10 ⁵/ mL is inoculated in the flat culture plate in 96 holes, every hole 100 μ l.Cultivate and abandon supernatant after 24 hours, each hole adds the nutrient solution 200 μ l that contain different concns PSP94 respectively, establishes 3 multiple holes for every group, and negative control is the bacterium periplasmic space supernatant purifying thing that only contains plasmid pBV220 of the same concentrations of the cultivation same period.After the cell cultures 44 hours, each hole adds freshly prepared 5 μ g/mLMTT respectively, 37 ℃ of incubations 4 hours, supernatant discarded, the DMSO that adds 100 μ l respectively continued to hatch 4 hours at 37 ℃, detected absorbancy (A) value at wavelength 570nm place on enzyme connection detector.Each organizes the inhibiting rate calculation formula: inhibiting rate (%)=(control group A mean value-experimental group A mean value)/control group A mean value * 100%.

By body outer cell proliferation experiment (mtt assay) with flow cytometer carries out dna content and cell cycle analysis shows, the secretor type PSP94 of reorganization has very strong biological activity, can produce the growth-inhibiting effect to prostate cancer PC-3 cell in the mode of time and dose-dependently, as Fig. 8 and shown in Figure 9, recombinant protein concentration 1000ug/ml effect 24 hours, inhibiting rate can reach more than 50%, acts on apoptosis occurring after 48 hours.Under the phase microscope, the cellular control unit adherent growth in order, mostly is fusiformis, is of moderate size, and kernel is clear, visible nuclear fission phase; PSP94 function cells quantity obviously reduces, and form is irregular, cell shrinkage, and particle increases, and cell debris increases (Figure 10).Above result shows that rshPSP94 has the activity that suppresses prostate cancer cell propagation.

(2) Flow Cytometry detects the influence of cell cycle: the PC-3 cell is divided into two groups, and experimental group adds heavy thin PSP94 albumen, and final concentration is 1 * 10 ^-4M, control group adds the bacterium periplasmic space supernatant product that contains empty plasmid of same concentrations.After the cell cultures 24 hours and 48 hours, make single cell suspension respectively,, calculate each cell cycle inner cell proportion, analysis of cells apoptosis situation through propidium iodide dyeing flow cytometry analysis.

Flow cytometry analysis PSP94 is to the influence of PC-3 cell cycle, the results are shown in Table 1, after the 10ug/ml function cells 24 hours, the cell cycle gets muddled, and shows as S, the minimizing of G2+M phase cell count, G1 phase cell count showed increased, act on after 48 hours, the increase of G1 phase cell count is more obvious, accounts for 85% of total cell count, show that the cell cycle is subjected to press down, mainly be suppressed at the G1 phase.Dna content distributes as shown in figure 11.Inferior 2 times of body peaks appear in front, G1 peak, show that apoptosis (during apoptosis, dna break generates little dna fragmentation, discharges in cell, causes inferior 2 times of body peaks to occur) appears in cell.

Table 1 reorganization PSP94 is to the influence of PC-3M cell cycle

??Groups	??G0/G1	G2/M	??S	??Apoptosis
					??Control	??47.99±2.3	5.35±0.9	??46.65±3.8	??0.64±0.2
??PSP94(24h)	??78.52±3.4▲▲	12.1±0.8▲	??9.70±0.6▲▲	??0.95±0.1▲
					??PSP94(48h)	??85.0±2.9▲▲	5.67±0.6	??9.33±0.7▲▲	??5.12±0.3▲▲

▲▲P＜0.01，▲P＜0.05?vs?Control

Claims (4)

1, the method for a kind of recombining human PSP 94 protein secreting, expressing in intestinal bacteria comprises the following steps:

One, the clonal expansion of PSP94cDNA

Extract the total RNA of prostata tissue, angle by the RT-PCR method and get Chinese PSP94cDNA sequence, insert among the cloning vector pUC19, carry out sequencing after the PCR scalping;

Two, the structure of recombinant secretor type expression vector

PSP94 protein coding gene behind the double digestion, signal peptide and pBV220 plasmid are connected construction recombination plasmid pBV-PSP94;

Three, reorganization PSP94 protein expression

Positive recombinant plasmid pBV-PSP94 is the transformed into escherichia coli bacterial strain respectively, as JM109, HB101 or LB21 bacterial strain, is 40 ℃ through groping protein induced expression optimum temperuture, and optimum expression time is 5 hours.

2, according to claim 1 in the method for recombining human PSP 94 protein secreting, expressing in intestinal bacteria, the step 2 signal peptide is taked the natural diphtheria toxin signal peptide of modified mistake.

3, in the method as recombining human PSP 94 protein secreting, expressing in intestinal bacteria as described in the claim 2, the natural diphtheria toxin signal peptide of the modified mistake of step 2 is:

ES1 is that the signal peptide that contains the EcoRI restriction enzyme site is had a mind to strand primer:

5’-CGGAATTCGTGAGCAGAAAACT-3’

ES2：5’-GATGC?ATG?CGC?TGA?AGG?TGG?GGC?CCC?TAT?CCCCAG?TAG?CGC?CCC?TAT?TAA?GAT?TGA?CGC?AAA?CAG?TTT?TCTGCT?CAC?GAATTCCG-3’

4, according to claim 1 in the method for recombining human PSP 94 protein secreting, expressing in intestinal bacteria, the JM109 bacterial classification that step 3 contains positive recombinant plasmid pBV-PSP94 and empty pBV220 plasmid is inoculated in 100ml respectively and contains in the LB liquid nutrient medium of ammonia benzyl, 31 ℃ of shaking culture, treat that bacterium liquid OD600 reaches at 0.6 o'clock, temperature is transferred to 40 ℃, continue to cultivate 5h.

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