CN101525615A - Defensins gene expressed in human epididymis, and cloning, expression and application of same - Google Patents

Abstract

The invention belongs to the fields of biotechnology and medicine science, and provides a nucleic acid sequence of a defensins gene HEL-75 specifically expressed in the human epididymis and an amino acid sequence for expressing protein. The invention also discloses a method for preparing HEL-75 protein by genetic engineering technology or chemical synthesis; the HEL-75 protein is a new member of a beta-defensin family and is specifically expressed in human epididymis and bonded with sperms to prompt that the protein is associated with the mature and defense function of sperms. The HEL-75 protein not only can be used in the preparation of medicaments for resisting bacterial, fungus, viruses and tumors, provides a novel molecular target for developing novel contraceptive medicine, carrying out aid diagnosis and treating infertility, and is favorable for researches on male reproductive function.

Description

The clonal expression of the phylaxin gene of people's epididymal expression and application

Technical field

The invention belongs to biotechnology and medical field.Specifically, the present invention relates to nucleotide sequence and the proteic aminoacid sequence of this genetic expression of a kind of phylaxin gene HEL-75 of people's epididymis specifically expressing, and this proteic preparation method and anti-infective, antitumor, develop the biological applications in new contraceptive bian and auxiliary diagnosis and the treatment infertility.

Background technology

The Mammals alexin is the congenital immunity factor of body, and it is the small molecule cationic antibacterial peptide.According to aminoacid sequence, six halfcystines to and the number difference of exon be divided into two big classes: α-alexin and beta-alexin.α-alexin is rich in arginine, is made up of 29-35 amino acid, has three pairs with C1-C6, C2-C4, the disulfide linkage that C3-C5 arranges.They mainly are expressed in neutrophilic leukocyte and the paneth's cell.Up to the present, five kinds of different people's α-phylaxin genes and six kinds of α-alexin proteins have been reported altogether.The Mammals beta-alexin also contains about 35 amino acid whose residues, and six halfcystines are with C1-C5, C2-C4, C3-C6 mode form three pairs of disulfide linkage different with α-alexin.More what is interesting is; in a plurality of tissues and cell type, express different with α-alexin; the Mammals beta-alexin tends to be expressed in the epithelial cell in male genetic road, particularly epididymis and the testis, they at tissue with providing the first pipe protection barrier for body between the environment.

Recently, by the genome bioinformatics method of utilization system, 42 and 39 beta-alexin genes and pseudogene from rat and people's genome, have been found respectively.They all form four to five gene clusters in karyomit(e).More curiously, mainly be expressed in respiratory tract and the upper digestive tract except Defb4 (people BD2 homologous gene) in the RT-PCR analysis revealed rat, all other beta-alexin gene all tends to be expressed in male genetic road, particularly epididymis with in the testis.And the beta-alexin that great majority are expressed in the reproductive tract is subjected to grow with androgenic regulation and control, and they are expressed in the sexual maturity process and are strengthened.These characteristics show that beta-alexin may play double base in reproduction and host defense.

Summary of the invention

The purpose of invention is from people's epididymis cDNA library (application number: 200510059634.5, open (bulletin) number: clone a new HEL-75 gene (number of registration: EF426755) CN1840544), the dna sequence dna of code book invention fusion rotein is provided, the recombinant expression vector of the dna sequence dna that carries code book invention fusion rotein is provided, by bioinformatic analysis the HEL-75 gene is carried out the biological function prediction, and its recombinant expression protein is carried out the biological function research of system.

One, technological line of the present invention

1. gene clone and sequential analysis

2. the structure of expression vector

3. reorganization HEL-75 proteic purifying

4. the proteic biological function research of reorganization HEL-75

Two, concrete steps

1. gene clone and sequential analysis

Obtain a clone from young adult's epididymis cDNA library that this research department makes up, the utilization electronic cloning has obtained full-length gene, called after HEL-75.Find that through BLAST comparison back the gene that is numbered NM_207469 in it and the ncbi database has 100% homology, belongs to the alexin family member.Utilize GENSCAN (http://genes.mit.edu/GENSCAN.html) to analyze the HEL-75 gene structure, chromosomal localization utilizes the BLAT instrument among the Genome Browser (http://genome.ucsc.edu/cgi-bin/hgBlat) to analyze.The signal peptide cutting site of HEL-75 proteins encoded is predicted with SignalP (http://www.cbs.dtu.dk/services/SignalP); N-glycosylation and phosphorylation site are predicted with ProfileScan: http://myhits.isb-sib.ch/cgi-bin/PFSCAN; The series comparison is analyzed with CLUATAL W: http://www.ebi.ac.uk/Tools/clustalw/index.html.

2. the structure of expression vector

According to the gene order of HEL-75, the special primer of the synthetic ripe coding region of a pair of amplification: upstream HEL75-F:5 '-TT GGTACCGACGACGACGACAAGGGTGGGTCAAAATGTGTG-3, downstream: HEL1-R:5 '-GGC GAATTCTCATGATGTTACGGTCGTTTGTTGC-3 '.Upstream and downstream is introduced restriction enzyme site KpnI and EcoRI respectively.With people's epididymis cDNA library is template, by the direct amplifying target genes of PCR, is cloned into the cloning vector pGM-Tvector evaluation of checking order then.Be cloned among the expression vector pET32b (+) by KpnI and EcoRI site through the HEL-75 gene after the order-checking evaluation, make its reading frame consistent with fusion tag.Recombinant expression vector pET32b (+)-hBD32 changes E.coliBL21 (DE3) competent cell over to, the gained engineering strain through 1mM IPTG 32 ℃ induce 4h after, all components separates with 15%SDS-PAGE, the expression of the bright blue G-250 staining analysis engineering bacteria of Kao Masi.All molecular cloning experiments are all carried out according to standard program.

The reorganization HEL-75 purifying

According to the His-Tag in the carrier, utilization " two step nickel affinity chromatography methods " is carried out separation and purification to reorganization HEL-75 albumen.Briefly, " the first step affinity chromatography " is used for purification of Recombinant Trx-HEL-75 fusion rotein.Fusion rotein recombinant enterokinase cracking then discharges fusion tag.At last, reorganization HEL-75 albumen is reclaimed in utilization " the second step affinity chromatography ".Recombinant protein behind the purifying carries out quantitatively preserving with lyophilize then with Bradford (Bradford 1976) method.

4.HEL-75 the biological function of recombinant protein research

According to the 26S Proteasome Structure and Function prediction of HEL-75 gene, reorganization HEL-75 albumen has been carried out following functional screening and evaluation: (1) rhHEL-75 anti-microbial activity detects; (2) expression analysis of HEL-75 gene in people's vital tissue, and HEL-75 albumen indirect immunofluorescence detects; (3) functional study of HEL-75 aspect sperm motility, fertilization and fetal development.

The present invention, screens with identifying the HEL-75 protein biology and learns function by at in-vitro recombination expression by above experimental procedure tentative prediction Human epididymis Li-1 gene structure and function.People HEL-75 gene is made up of two exons, is positioned at human chromosome 20p13 position, 95 amino acid of total length coding, and 22 amino acid of N-end are signal peptide.HEL-75 albumen has six cysteine residues, and has a plurality of potential posttranslational modifications site, analyzes to belong to the beta-alexin family member.The mRNA of this gene has the expression of par in people's caput epididymidis, body, portion and lung, but proteins encoded is only expressed at adult's caput epididymidis, body and afterbody, and the strongest at the epididymis head expression, afterbody is the most weak, does not then detect proteic expression in lung.In addition, HEL-75 albumen can specific combination in sperm, but in testis and intratesticular sperm, do not detect this albumen, show HEL-75 be in epididymis with the sperm bonded.Reorganization HEL-75 albumen is inhibited to intestinal bacteria, and this effect has concentration and time-dependent manner.Experiment in vitro fertilization shows that HEL-75 does not mediate smart ovum fusion process.

Positively effect of the present invention specifically describes as follows:

Utilize bioinformatics method, obtain a clone from people's epididymis cDNA library that this research department makes up, the utilization electronic cloning has obtained full-length gene, and called after HEL-75 belongs to the epididymis specific expression gene.Newfound people HEL-75 gene cDNA total length 2121bp, 95 amino acid of encoding, molecular weight of albumen is 10.6kDa.Wherein, 22 amino acid of N-end are signal peptide, and mature peptide comprises 73 amino acid, and theoretical iso-electric point is 9.62.People HEL-75 gene has six conserved cysteine residue.HEL-75 albumen exists a N-tetradecanoyl site (G23GSKCV) and a N-glycosylation site (N50ASR).In addition, also may there be four phosphorylation sites, comprise a cAMP-and cGMP-dependent protein kinase phosphorylation site (R79RNT) and three protein kinase C phosphorylation sites (S52RK, S77RR and T82QR).And, in molecule, also found a potential coagulation factors V LSPD tumor-necrosis factor glycoproteins (L62PKPDLPQL).Adopt sxemiquantitative RT-PCR to analyze the HEL-75 gene people's caput epididymidis, body, tail, testis, the heart, liver, spleen, lung, kidney, differential expression in the tissue such as stomach, the mRNA of this gene does not express in other tissue substantially in the expression that people's caput epididymidis, body, portion and lung have par.But proteins encoded is only expressed at adult's caput epididymidis, body and afterbody, and the strongest at the epididymis head expression, and afterbody is the most weak, does not then detect proteic expression in lung.

The clone of HEL-75 gene and the method for recombinant expressed and purifying have been set up.

Find that HEL-75 albumen helps congenital host defense.HEL-75 albumen belongs to the beta-alexin family member, and has anti-microbial activity.Experiment finds that reorganization HEL-75 albumen is inhibited to intestinal bacteria, this effect has concentration and time-dependent manner, illustrate that HEL-75 albumen may have immunocompetence as a kind of autarcetic medium in body, play an important role in having improved the host defense function.For the anti-microbial activity research in vivo of HEL-75 albumen is laid a good foundation.This alexin with anti-microbial activity provides new source for the research and development new antibiotic.

HEL-75 albumen not only has provide protection to sperm as specific expressed alexin in reproductive tract, also has the sophisticated functions that participates in other physiological processs.Experiment finds that HEL-75 can combine with sperm.The expression of most of beta-alexin is regulated by male sex hormone in the arrenotoky road of people and mouse, promptly in expression amount increase in sexual maturing period; The variation of androgenic adjusting level also may be regulated and control HEL-75 albumen.The present invention with the adult man epididymis as experiment material, from HEL-75 gene mRNA and HEL-75 albumen the expression analysis tissue as seen, sexual maturity stage HEL-75 albumen exists obviously expresses.Sophisticated process may rely on the proteic expression of HEL-75 in the sperm epididymis, and by in conjunction with HEL-75 albumen, further regulates and control spermioteleosis.Experimental result of the present invention shows, fails to influence significantly normal fertilization and early embryonic development through the sperm group after the sealing of HEL-75 protein antibodies.Further illustrating HEL-75 albumen plays a role to the sperm in epididymis stage of maturity.

Description of drawings:

Fig. 1: (A) people HEL-75 gene cDNA and corresponding amino acid sequence.HEL-75 gene ORF (open readingframe) contains 288bp, 95 amino acid of encoding, and wherein 22 amino acid of N-end are signal peptide (italic).Tetradecanoyl site (G23GSKCV), N-glycosylation site (N50ASR) and coagulation factors V LSPD tumor-necrosis factor glycoproteins (L62PKPDLPQL) are represented with circle, square and double bracket respectively.CAMP-and cGMP-dependent protein kinase phosphorylation site (R79RNT) and protein kinase C phosphorylation site (S52RK, S77RR and T82QR) are used respectively

"-" expression.(B) HEL-75 gene structure.The HEL-75 gene is made up of two exons (square frame), and there is the intron (straight line) of a long 1240bp centre.Black part is divided into the protein-coding region exon, and white portion is the non-coding region exon.

Fig. 2: the strategy by amalgamation and expression prepares recombinant protein (Fig. 2 A).Mainly there be (Fig. 2 B) in SDS-PAGE analysis revealed, fusion rotein Trx-HEL-75 with soluble form.Bigger (Fig. 2 C) of natural HEL-75 molecular ratio reorganization.Diagram: (A) HEL-75 intestinal bacteria amalgamation and expression protein structure figure. (B) expressing fusion protein analysis: 1,3,5: three BL21 (DE3)/pET32b (+)-HEL-75 clone's indissolvable component; 2,4,6: the soluble constituent that these clones are corresponding. (C) polyclonal antibody specificity analyses: 1, the Trx-HEL-75 fusion rotein of purifying; 2, the rHEL-75 albumen of purifying; 3, adult's epididymal fluid extract.

Fig. 3: sxemiquantitative RT-PCR analyzes the HEL-75 gene 1, caput epididymidis; 2, body of epididymis; 3, cauda epididymidis; 4, heart; 5, lung; 6, liver; 7, spleen; 8, kidney; 9, stomach; 10, the differential expression in the tissue such as testis, the mRNA of this gene does not express in other tissue substantially in the expression that people's caput epididymidis, body, portion and lung have par.House-keeping gene β-actin is confidential reference items.

Fig. 4: people HEL-75 is expressed in lung and other tissue less than detecting but the proteic expression of HEL-75 at head, body and the afterbody of adult's epididymis.Diagram: A row, caput epididymidis negative control; B row, caput epididymidis; C row, body of epididymis; D row, tail; E row, testis .1 row, sample differ figure; 2 row, nucleus dyeing (PI); 3 row, HEL-75 dye (FITC mark); 4 row, the 2nd row and the 3rd row overlay chart.(100×).

Fig. 5: the indirect IF staining analysis confirms that the HEL-75 protein binding is (100 *) .A row in whole sperm, HEL-75 albumen; B row, positive control protein B D118; C row, negative control .1 row, sample differ figure; 2 row, nucleus dyeing (PI); 3 row, HEL-75 dye (FITC mark); 4 row, the 2nd row and the 3rd row overlay chart.

Fig. 6: the empty host bacterium of transform bacteria growth analysis (A). (B) transform empty carrier host bacterium. (C) transform the HEL-75 gene. begin to survey OD (600nm) from t=0 every the 1h sample thief. when bacterium arrives exponential phase, each sample is got half culture, and adding final concentration is 0.4mM IPTG.Survey the OD value every the 1h sampling then, each value is surveyed three times, averages then.Real quadrangle does not contain IPTG; Empty trilateral is after IPTG induces.

Fig. 7: reorganization HEL-75 albumen is inhibited to the Gram-negative intestinal bacteria, and this effect has concentration and time-dependent manner (Fig. 7).Bacterium is followed 12.5g/ml (■) respectively in the time of 37 ℃; 25g/ml (●); 50g/ml (▲) and 100g/ml

RHEL-75 cultivates 15～120min altogether.Be coated with flat board after the different time sampling dilution, calculate the clone who forms.

Fig. 8: experiment in vitro fertilization shows, still can normal fertilization through the sperm after the antibody sealing, and also fertilization efficient compares constantly substantially with control group, and development of fertilized ova is normal.A-B: antibody sealing people sperm immunization fluorescence proof diagram, A is an experimental group; B is a control group.C-D: (IVF) in vitro fertilization back fetal development situation.C is an experimental group; D is a control group.1, the time of 3,5 these fetal developments of expression.Magnification: 200; Scale: 50 μ m.

Specific implementation method

Below by embodiment the present invention is specifically described; be necessary to be pointed out that at this present embodiment only is used for the present invention is further detailed; can not be interpreted as limiting the scope of the invention, the person skilled in the art of this area can make some nonessential improvement and adjustment according to the content of the invention described above.

Embodiment 1HEL-75 gene clone and prokaryotic expression thereof

1 experiment material

1.1 bacterial strain and plasmid

Bacterial strain or plasmid explanation source

Preserve in this laboratory of the conventional cloning host bacterium of E.coli DH5 α

The conventional expressive host bacterium of E.coli BL21 (DE3) is available from Novagen company

PMD-18T Vector cloning vector has the Ampr gene and purchases the company in TaKaRa

PET-32b (+) expression vector has the Ampr gene available from Novagen company

1.2 main biochemical reagents

All restriction enzymes, polysaccharase and T4DNA ligase are available from Dalian Takara company.Yeast leaches cream and peptone is that Oxoid company is original-pack, Tutofusin tris (Tris) is the packing of the magnificent company in Canada production Beijing, sodium laurylsulfonate (SDS), ethidium bromide (EB), penbritin (Amp) and that penicillin of card (Kan) are Sigma company and produce, and other conventional chemical reagent are homemade analytical pure.

2 experimental techniques

2.1 design of primers is with synthetic

Designed a pair of special primer according to coding HEL-75 gene mature protein coding sequence:

Upstream HEL75-F:5 ' TTGGTACCGACGACGACGACAAGGGTGGGTCAAAATGTGTG-3,

Downstream HEL75-R:5 ' GGCGAATTCTCATGATGTTACGGTCGTTTGTTGC-3 ', upstream and downstream is introduced restriction enzyme site KpnI and EcoRI respectively.Primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.

2.2HEL-75 the clone of gene

2.2.1PCR amplification HEL-75 gene

Application of sample: in 0.5ml PCR pipe according to following amplification system application of sample:

ddH2O 37.5l

10PCR?buffer 5l

Taq enzyme (10U/l) 0.5l

dNTPs(2.5mM) 4l

HEL-75F(25M) 1l

HEL-75R(25M) 1l

CDNA template 1l

Cumulative volume 50.0l

Amplification: the 2400PCR instrument that places PE company to produce the PCR pipe increases, and amplification program is: 94 ℃ of pre-sex change 5min; (94 ℃, 30s; 60 ℃, 40s; 72 ℃, 90s) 30 circulations; 72 ℃ of insulation 7min; 4 ℃ of preservations

Detect: get 2 μ l products after reaction finishes and detect with 1.2% agarose gel electrophoresis.

2.2.2PCR amplified production reclaims

Utilize the Wizard DNA clean-Up System of Promega company, reclaim the PCR product from sepharose.Get 1 μ l and carry out agarose gel electrophoresis estimation recovery production concentration, all the other are put-20 ℃ of refrigerators and preserve standby.

2.3 the structure of cloning vector and evaluation

2.3.1PCR the recovery product is connected with cloning vector

Add the 0.5ml centrifuge tube successively by following dosage, abundant mixing with whizzer centrifugal a moment, is received the pipe end with the tube wall drop.Place 4 ℃ of refrigerators to connect 16-18h.

Solution?I 5μl

T-carrier 1 μ l

HEL-75 gene 4 μ l

Cumulative volume 10 μ l

2.3.2 connect product transformed competence colibacillus cell

Get 0.1mol/l CaCl2 33.6 μ l and 1 * SSC, 16.8 μ l solution in the 1.5ml centrifuge tube, mixing adds and connects product 10 μ l, fully ice bath 1.5min behind the mixing;

Add competent cell 100 μ l, mixing gently, ice bath 5-10min;

42 ℃ of static water-bath heat shock 2min put cooled on ice 1min then at once;

Add 850 μ l LB liquid nutrient mediums, 37 ℃, the 200r/min shaking table is cultivated 1h;

5000r/min, the centrifugal 2min of normal temperature collects thalline, and it is resuspended to add the fresh LB substratum of 200 μ l;

Get 100 μ l conversion fluids and coat (30min is coated with 16 μ l 10mg/mlX-Gal and 4 μ l 200mg/ml IPTG mixed solutions in advance) on the LB culture medium flat plate that contains 100mg/ml Amp, carry out blue hickie screening;

37 ℃ of constant temperature culture 12-16h;

2.3.3 the extraction of recombinant plasmid

Choose white single colony inoculation to containing in the corresponding antibiotic LB liquid nutrient medium from transform flat board, in 37 ℃, the 300r/min shaking table is cultivated 16-18h;

Get 1.5ml bacterium liquid in the 2ml centrifuge tube, 8000r/min, 4 ℃ of centrifugal 2min;

Remove supernatant fully, add the Solution I of 150 μ l precoolings, vortex vibration suspension thalline;

Add 300 μ l and newly prepare Solution II, be inverted mixing gently, room temperature leaves standstill 5min;

The Solution III that adds 200 μ l precoolings, mixing postposition 5-10min on ice gently;

10000r/min, 4 ℃ of centrifugal 10min.Get supernatant in a new centrifuge tube, add isopyknic Virahol, room temperature is placed more than the 10min behind the mixing;

10000r/min, the centrifugal 10min of room temperature removes supernatant.Wash precipitation 2-3 time with 70% ethanol of 1ml precooling, at last air-dry precipitation in super clean bench;

With 20 μ l TE (pH8.0) dissolution precipitations, add again RNase A to final concentration be 20 μ g/ml, in about 37 ℃ of incubation 60min, to remove RNA;

Plasmid is preserved standby in 4 ℃ of refrigerators;

2.3.4 the enzyme of recombinant plasmid is cut evaluation

Application of sample: Eco RI/Kpn I double digestion reaction system:

10×buffer?M 2.5μl

Kpn?I(10U/μl) 0.5μl

EcoR?I(10U/μl) 0.5μl

Recombinant plasmid 4 μ l

ddH2O 17.25μl

Cumulative volume 25 μ l

Enzyme is cut: fully centrifugal a moment behind the mixing, place 37 ℃ of water-bath enzymes to cut more than the 3h again

Detect: cut product, screening positive clone with 1.2% agarose gel electrophoresis enzyme analysis.

2.3.5 the sequential analysis of recombinant plasmid: the picking positive colony, by precious biotech firm sequencing analysis;

2.4 the structure of expression vector and evaluation

2.4.1HEL-75 the preparation of gene

The extraction of pMD18T-HEL-75 plasmid: the method for pressing the 2.3.3 joint is extracted plasmid;

The enzyme of pMD18T-HEL-75 plasmid is cut: do enzyme by the method for 2.3.4 joint and cut;

The recovery of HEL-75 gene: the method recovery enzyme by the 2.2.2 joint is cut product.

2.4.2 the preparation of expression vector pET32b (+)

The extraction of pET32b (+) plasmid: the method for pressing the 2.3.3 joint is extracted plasmid;

The enzyme of pET32b (+) plasmid is cut: do enzyme by the method for 2.3.4 joint and cut;

The big segmental recovery of pET32b (+) carrier: the method recovery enzyme by the 2.2.2 joint is cut product.

2.4.3HEL-75 gene is connected with expression vector

Application of sample: add successively at the 0.5ml centrifuge tube:

ddH2O 4μl

10 * ligase enzyme buffer, 1 μ l

PET32b (+) carrier 1l

HEL-75 gene 3 μ l

T4-DNA ligase enzyme (3U/ μ l) 1 μ l

Cumulative volume 10 μ l

Abundant mixing with whizzer centrifugal a moment, is regained the pipe end with the tube wall drop.

Connect: place 16 ℃ of water-baths to connect more than the 20h.

2.4.4 connect the conversion and the evaluation of product

Transform: connect the method transformed competence colibacillus cell that product provides according to the 2.3.2 joint

Identify: the method for extracting plasmid, 1.2.3.4 joint according to the method for 2.3.3 joint is done enzyme and is cut evaluation

2.5 structure and the screening of engineering bacteria BL21 (DE3)/pET-32b (+)-HEL-75

The preparation of (2.5.1pET-32b+)-HEL-75 plasmid

The extraction of pET-32b (+)-HEL-75 plasmid: the method according to chapter 1 2.4.4 joint is extracted plasmid;

Plasmid purification: in above-mentioned plasmid, add the 13%PEG8000 solution that equal-volume contains 1.6M NaCl, mixing, room temperature is placed 5min, then in 12000r/min, the centrifugal 5min of room temperature; Remove supernatant, precipitation is dissolved in earlier among the 500 μ lTE (pH8.0), adds equal-volume phenol then: chloroform: primary isoamyl alcohol (25: 24: 1), fully mixing; 12000r/min, the centrifugal 5min of room temperature gets supernatant then in a new centrifuge tube, adds the equal-volume chloroform: primary isoamyl alcohol (24: 1), fully mixing; 12000r/min, the centrifugal 5min of room temperature gets supernatant then in a new centrifuge tube, adds NaAc (pH5.2) solution of 1/10 volume 3M and the dehydrated alcohol of 2 times of volume precoolings, fully in the mixing postposition-20 ℃ refrigerator about 15min; 12,000r/m, 4 ℃ of centrifugal 10min abandon supernatant.70% washing with alcohol with the 1ml precooling precipitates 2-3 time, and is air-dry in super clean bench then; DNA behind the purifying is dissolved among the 20 μ l TE (pH8.0), gets 2 μ l electrophoresis detection purity and estimated concentrations, remaining put in-20 ℃ of refrigerators preserve standby;

2.5.2E.coli the conversion of BL21 (DE3)

Transform: the method transformed competence colibacillus cell that pET-32b (+)-HEL-75 plasmid provides according to the 1.2.4.3 joint.

2.5.3 bacterium colony PCR screening positive clone

Application of sample: in 0.5ml PCR pipe according to following amplification system application of sample:

ddH2O 19.5μl

10×PCR?buffer 2.5μl

dNTPs(2.5mM) 2μl

HEL-75F(25μM) 0.5μl

HEL-75R(25μM) 0.5μl

Cumulative volume 25 μ l

Choose single bacterium colony with the sterilization toothpick in above-mentioned PCR pipe from transforming flat board, stir gently and make the thalline dissolving.Attention performs mark by reference numeral on flat board.

Cracking: the PCR pipe is put on the PCR instrument, established 99 ℃ of heating 5min, with lysing cell

Take out the PCR pipe, every pipe adds 0.25 μ l Taq enzyme (10U/ μ l)

Amplification: place the PCR instrument to increase again the PCR pipe by following program:

94 ℃ of pre-sex change 5min; (94 ℃, 30s; 60 ℃, 40s; 72 ℃, 90s) 30 circulations; 72 ℃ of insulation 7min; 4 ℃ of preservations;

Detect: get 2 μ l products after reaction finishes and detect, with screening positive clone with 1.2% agarose gel electrophoresis;

2.6HEL-75 the abduction delivering of gene

A single colony inoculation that contains recombinant plasmid is contained in the suitable antibiotic LB liquid nutrient medium, in 37 ℃ of shaking table overnight incubation in 5ml;

Draw 0.2ml incubated overnight liquid and contain in the suitable antibiotic fresh LB substratum, be cultured to the OD600 value between 0.8-1.0 in 37 ℃ of shaking tables in 10ml;

Above-mentioned bacterium liquid is divided into two parts, and a adding 100mM IPTG is 1mmol/l to final concentration, not inductive contrast of another part conduct.Continue to cultivate 4h in 37 ℃;

Respectively get the 1ml culture in the 1.5ml centrifuge tube, 5000r/min, the centrifugal 5min of room temperature collects thalline;

Thalline is resuspended in 100 μ l, 1 * sds gel sample loading buffer, then in 100 ℃ of heat denatured 3min.Sample after the sex change is stored in 4 ℃, analyzes in order to being SDS-PAGE;

2.7 recombinant protein SDS-PAGE analyzes

By specification is installed the electrophoresis sheet glass

Glue: adopt 12% separation gel and 5% to concentrate glue, it is filled a prescription as following table:

Each component title 12% separation gel (10ml) 5% concentrates glue (3ml)

ddH2O 3.3 2.1

30％Acrylamide 4.0 0.5

1.5M?Tris-HCl(pH8.8) 2.5 0

1.0M?Tris-HCl(pH6.8) 0 0.38

10％SDS 0.1 0.03

10% Ammonium Persulfate 98.5 0.1 0.03

TEMED 0.004 0.003

Prepare 12% separation gel 10ml by last table, in the gap of two sheet glass, pour into separation gel rapidly after adding TEMED, the volume of the separation gel of irritating account for the monoblock colloid long-pending 2/3rds.On separation gel, cover one deck ddH2O carefully with suction pipe, then gel vertically is positioned over natural coagulation under the room temperature.The separation gel polymerization is back (about 30min) fully, and inclining tectum ddH2O, and filter paper blots residual liquid again.By the concentrated glue 5ml of last table preparation 5%, rapidly pouring into concentrated glue on the polymeric separation gel, insert clean comb immediately behind the adding TEMED, carefully avoid bringing into bubble, then gel vertically is positioned under the room temperature.

Last sample: after concentrating glue polymerization fully, the careful comb that takes out, immediately with the deionized water wash loading slot to remove unpolymerized acrylamide, gel sets in electrophoresis apparatus, groove respectively adds Tris-glycine electrophoretic buffer up and down, gets rid of the bubble between the sheet glass.Add an amount of sample with microsyringe by predetermined order.

Electrophoresis: electrophoresis chamber is connected with power supply, and earlier the voltage with 80V carries out electrophoresis, treat that the tetrabromophenol sulfonphthalein forward position enters into separation gel after, again voltage is brought up to 150V, arrive separation gel bottom, powered-down then until tetrabromophenol sulfonphthalein.

Coomassie brilliant blue R250 dyeing:

(1) staining fluid: at 90ml methyl alcohol: water (1: 1, dissolving 0.25g Xylene Brilliant Cyanine G v/v) and in the mixed solution of 10ml glacial acetic acid, with the filter paper filtering dye liquor to remove particulate material;

(2) dyeing: unload lower glass plate from electrophoresis apparatus, carefully take out gel, and cut the orientation of one jiao of one mark gel in bottom near sample groove on the first left.Soak gel with an amount of staining fluid, be placed on the shaking table and dye about 2h in room temperature;

(3) decolouring: after dyeing finishes, reclaim staining fluid to reuse.Gel is soaked in the methyl alcohol-acetic acid solution that does not add dyestuff, is placed on the shaking table, change destainer 3-4 time therebetween up to distinguishing protein band in room temperature decolouring 4-8h.

(4) preserve: after the decolouring fully, gel is stored in the water that contains 20% glycerine, or uses 50% alcohol immersion earlier, treat to make when gel no longer dwindles dried glue and preserve.Be to keep HC hard copy, preferably to the preservation of taking pictures of stained gel.

2.8 recombinant protein soluble analysis

Induce: engineering bacteria is induced according to the method for 1.2.6

Broken: the bacterium liquid after getting 10ml and inducing, the centrifugal 5min of 5000r/min normal temperature, it is resuspended with 1ml 50mmol/L PBS (pH7.4) to collect thalline, uses the Ultrasonic Cell Disruptor smudge cells then.Adopt power level 4-5, the 40-50% energy rate, the 15-20 pulse is at rotation, ultrasonication 5min on ice.With the centrifugal 10min of 12000r/min, get supernatant and newly manage in one after the cytoclasis, precipitation then uses 1ml 50mmol/L PBS (pH7.4) resuspended.Last solvablely cleer and peaceful precipitation after resuspended and respectively get 50 μ l, add 50 μ l2 * sds gel sample-loading buffer, 100 ℃ of heat denatured 3min.The SDS-PAGE electrophoresis is carried out in sampling, to analyze the existence of expression product.

SDS-PAGE analyzes: the method according to 2.7 is carried out

3. result and analysis

3.1HEL-75 gene clone

By the direct amplification HEL-75 gene from normal young people epididymis cDNA library of PCR.The result is presented at the 250bp place a specific band, identical with the ripe coding region of the HEL-75 that intends amplification size.

3.2 recombinant strain screening

Recombinant expression vector pET32b (+)-HEL-75 transforms BL21 (DE3) competent cell, and the bacterium colony pcr analysis shows that selected clone is positive.The gained engineering strain is induced 4h with 1mM IPTG at 32 ℃, uses the SDS-PAGE electrophoresis then, the Xylene Brilliant Cyanine G R-250 detection of dyeing.As seen have one significantly to induce band to produce, and fusion rotein exist with inclusion body and two kinds of forms of solubility.Above result shows, external the HEL-75 gene has been carried out successful reorganization.

The proteic purifying of embodiment 2rhHEL-75

1. engineering bacteria activation:

Get glycerine and guarantee that the engineering bacteria of depositing is inoculated in 30ml and contains in the LB nutrient solution of penbritin (50ug/ml), 37 ℃, the 200rpm shaking table is cultured to mid-log phase (A550=0.5-1.0).

2. engineering bacteria is dull and stereotyped cultivates:

It is streak culture in the LB culture plate that contains penbritin (50ug/ml) to get the activatory engineering bacteria, has the flat board sealing of single bacterium colony to preserve with long.

3. engineering bacteria large scale culturing, the great expression target protein:

Picking engineering bacteria colony inoculation contains in the LB nutrient solution of penbritin (50ug/ml) in 50ml, 37 ℃ of incubated overnight.Get the 20ml overnight culture and insert the LB nutrient solution that 500ml contains penbritin (50ug/ml), 37 ℃ of shaking tables are cultured to mid-log phase (A550=0.5-1.0).Adding IPTG is 1mmol/L to final concentration, 37 ℃ of cultivation abduction delivering 4h.Abduction delivering finishes, and collects thalline in 4 ℃ with the centrifugal 10min of 6000g.

4. inclusion body preparation, washing and dissolving:

With wet thallus multigelation 3 times, add 30ml lysis buffer (50mmol/L Tris-Cl, pH8.0,1mmol/L EDTA, 100mmol/L NaCl) and hanged thalline.Carrying out ultrasonic bacteria breaking under the condition of ice bath, 2min/ time, totally 7 times.11000g, 4 ℃ of centrifugal 15min get precipitation.Precipitation is resuspended in the 0.1mol/L Tris (pH8.0) of 2mol/L urea.11000g, 4 ℃ of centrifugal 15min get precipitation.8mol/L urea, 0.1mol/L Tris (pH8.0) sex change liquid dissolving inclusion body adds DTT to 5mmol/L final concentration.Room temperature sex change 40min.

20000g, 14 ℃ of centrifugal 20min get supernatant.

5. target protein on-column refolding and purifying simultaneously:

8mol/L urea, 0.1mol/L Tris (pH8.0), 0.5mol/L NaCl damping fluid balance Chelating SepharoseFF affinity column.Sample affinity column on the centrifugal supernatant, the damping fluid flushing.50mmol/L imidazoles denatured state is the washing affinity column down.50mmol/LTris (pH8.0), 3 column volumes of 0.5mol/L NaCl damping fluid flushing affinity column.Target protein on-column refolding 24h.200mmol/L imidazoles wash-out target protein is collected elution samples.Ordinary method is carried out manipulation of regeneration to affinity column.

6. target protein detects:

Show target protein renaturation and purification effect by reduction and non-reduced SDS-PAGE electrophoresis detection, target protein purity 〉=93%, target protein monomer rate answers 〉=70%.

Embodiment 3 protein extracts and Western blot

People's epididymis total protein extract carries out according to document.Each sample total protein 20 μ g separates with 15%SDS-PAGE, and wet then forwarding to carried out western blot on the pvdf membrane.Mouse-anti people HEL-75 is how anti-to be one anti-(1: 5000), and two anti-ly are HRP-mark sheep anti-mouse igg (1: 500).The activity of horseradish peroxidase is analyzed with enhancement type HRP-DAB substrate colouring reagents box.

The preparation and the histogenic immunity fluorescence location of embodiment 4 anti-HEL-75 polyclonal antiserums (how anti-)

Resist prepares with BALB/C mice more.Briefly, every mouse was injected the complete freund adjuvant (CFA) of 50 μ g recombinant proteins and equivalent in the 1st day.Injected incomplete freund adjuvant (IFA) booster immunization of 25 μ g recombinant proteins and equivalent then at the 15th, 30 and 45 day.Get blood from eyeball on the 60th day, and analyzed tiring and specificity of antibody with ELASA and western blot behind the separation of serum.

The immunofluorescence dyeing method is seen document.Wherein one anti-is mouse-anti rHEL-75 how anti-(1: 400), and two anti-ly are FITC-mark sheep anti-mouse igg (1: 200), and nucleus dyes with PI.Sections all after the dyeing are used Laser Scanning Confocal Microscope (Leica TCS SP2AOBS) observations then with 80% glycerine mounting.Experiment is anti-as negative control with blood serum substituting before the mouse immune one simultaneously.

Embodiment 5 sperm immunization fluorescence location

Collect and coat on the slide glass of 1% gelatin bag quilt after sperm washs with PBS, dry naturally, fix 10 minutes with methyl alcohol then.The slide glass that contains sperm at room temperature sealed 1 hour with 3%BSA, added mouse-anti rhBD32 then and resisted (1: 200) more, and 4 ℃ are spent the night.Mice serum is done negative control before the immunity.With PBST (PBS containing 0.1%Tween-20) washing three times, add corresponding FITC-mark sheep anti-mouse igg two anti-(1: 200).Slide glass is used 80% glycerine mounting then with PBST washing three times.With Olympus BX-52 microscopic examination result.

Embodiment 6 bacterial growth analyses

Analysis induces Recombinant Protein Expression whether to suppress the growth of host bacterium in intestinal bacteria.Briefly, contain the bacterium incubated overnight of recombinant vectors or empty carrier after, with the LB substratum according to inoculation in 1: 40.At this moment, OD600 is surveyed in sampling, and sampling in every later on each hour is observed.When arriving exponential phase of growth, bacterium is divided into two parts.It is 0.1mM to final concentration that a copy of it adds IPTG, and another part do not add in contrast.Every later on OD600 that measured separately in each hour, each parallel replicate measurement three times.As negative control, blank host bacterium BL21 (DE3) is used to detect the toxicity of IPTG.

Embodiment 7 anti-microbial activity analyses

Utilization clone's colony-forming unit (CFU) is analyzed anti-microbial activity.Briefly, the E.coli XL-1blue of incubated overnight grows into 10mM PBS (pH 7.4) dilution of logarithmic phase (OD600=0.4-0.5) back.About 2 * 106CFU/ml bacterium mixes at the rhBD118 with 12.5～100 μ g/ml, 37 ℃ of cultivations.After beginning to cultivate 15,30,60and 120min is respectively in sampling.Sample is done serial dilution with 10mM PBS (pH 7.4), and each extent of dilution is got 100 μ l and is coated the LB agar plate, and 37 ℃ of overnight incubation are to forming single bacterium colony.Statistics bacterium colony number, anti-microbial activity represents that with the percentage ratio of bacteria living formula is: % survival=(albumen handle back survival clone number/handle clone's number of surviving in the back without albumen) * 100.Experiment replaces albumen to handle bacterium as negative control with 10mM PBS (pH 7.4).

Embodiment 8 sperm motilities and analysis in vitro fertilization

Utilize HEL-75 to resist sealing normal people sperm more, carry out sperm motility and analysis in vitro fertilization then.Experiment is carried out according to the method that vitrolife standard reagent box provides.Briefly, sperm uses SpermRinse-30 in 37 ℃, 5%CO earlier ₂Cultivate capacitation in 1 hour.Added mouse-anti rHEL-75 according to 1: 200 then and resist more, in 37 ℃, 6%CO ₂Cultivate and sealed in 2 hours.Sperm separated into two parts after the sealing, a part directly utilize computer aided system to analyze the mobility of sperm.Another part is with G-Fert washing 2 times, and to be adjusted to final concentration be 1.0 * 10 ⁷Cells/ml.Add 100 μ l sample of sperm in the culture dish of 35mm, cover paraffin oil then and put into 37 ℃, 6%CO ₂With stand-by in the saturated humidity incubator.Handle the after ripening ovocyte through G-MOPS and add during preprepared fertilization drips, at 37 ℃, 6%CO ₂Observe the fertilization situation with cultivating in the saturated humidity incubator, write down the fetal development situation every day.More than all the experiment use simultaneously the immunity before mice serum as negative control.

Sequence table

1, general information

(i) denomination of invention: the phylaxin gene of people's epididymal expression and clonal expression and application

(ii) sequence number: 2

2, the information of SEQ ID NO:1:

(i) sequence signature

(A) length: 2101

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: linearity

(ii) molecule type: cDNA

(xi) sequence description: SEQ ID NO:1:

ctccattaaa?ccaccaccag?ctccccaagc?caccccttca?gccatgaagt?tcctgctcct 1

ggtcttggca?gccctcggat?tcctgaccca?ggtgatccca?gccagtgcag?gtgggtcaaa 61

atgtgtgagt?aacaccccag?gatactgcag?gacatgttgc?cactgggggg?agacagcatt 121

gttcatgtgc?aacgcttcca?gaaaatgctg?catcagctac?tccttcctgc?cgaagcctga 181

cctaccacag?ctcatcggta?accactggca?atcaaggaga?agaaacacac?aaaggaaaga 241

caagaagcaa?caaacgaccg?taacatcata?ataaccactg?ctatcgcctc?caccaactca 301

gagaaatatc?atttccacag?ttccaattcc?tcctacattg?ctgagtacta?gccaaggctc 361

ctctttatgg?ggcagatatc?tatagccaac?cccaaaactt?ctgtcttcta?tcattctgtc 421

attcatctag?taactaattt?ggagtttgta?tctatcttac?gagaacaatc?atcatgcaga 481

ttcgtccaca?ggggatctgt?cagtttgggt?cctccaaatg?aaaaatgtca?agacagaatt 541

ggacatgcaa?aagattgact?gggagaacac?acctctgatg?gacaaaggtg?agacagagca 601

gccacaggca?gggagagcct?tcagactgca?acgctggcct?gatacgtgtc?aaaggagaga 661

gggatagagg?aggattgaat?agaaggagac?taagactgca?gctctaagaa?agtctcagcc 721

aaacagatgg?ggaggcccaa?agcaaggctt?gcccctcaga?ggagctcacg?cagggcagga 781

atagccaggt?tctcatatcc?caggggttca?gacttggctg?agaacagccc?ctggagaaca 841

tggggtgact?gctaccatag?gtctggaagt?atgaggctgt?ccaccaacta?tccccttgaa 901

gcaagttctc?ttgaaaggaa?atctaaacag?tgcaccccca?tggctgccac?ggagtataag 961

gagggagaga?aaggagctga?aagtctaggt?ttggccagct?aggtagactg?acttgtgagg?1021

tatttattta?ttcatttgag?taacaaagca?gacagaatac?atagccacca?ttggtagtac?1081

accccaaaag?caaggatggc?atgatgctgg?tgactcaaac?gtgcctactc?atggtgtcaa?1141

attggcataa?tcctcttggg?aagctgtgtg?gaaataagca?cagagaagca?gaactctaat?1201

tgcttaatcc?actaaacatt?acttctggga?attggctcat?cataaattat?ccaagagaaa?1261

gcacaaagtt?atgggcacaa?aggttttcca?tataatatta?tttaaaatgc?tgagaaaatg?1321

aaaaaatcta?aatggtgaaa?tatatactaa?tgccatctat?aaatacaaac?aaatagaatg?1381

tttatagaat?aatggaacat?aataacatta?ttcaaaattg?catttatgct?atagttgtca?1441

aaattgtctc?cttatatgat?acaaaactca?tgaaaattat?gacttttttg?tttggttgga?1501

aagcagaatt?atgcataaat?ttcctcttac?agttcgatgc?ccattagttt?tatataacat?1561

ttatttgaca?cgtactgact?tctatctgag?aagaacaaac?caaaacactc?aggcctaaat?1621

aattaaaaac?ggtcctaaaa?actagcaaac?cagataagaa?aagatgttaa?tgcccattcc?1681

ctaacttatg?tcttagacca?aaattaattc?tagatggttt?taaaatgaca?gtgtaaaagt?1741

aaagtattaa?aagattgtgt?ggtcaaatat?tcaatttaag?agcaaggaaa?ttcttataaa?1801

tataacaata?gaggcagaac?tcatgtaaga?ataaattgat?taggtggtat?taaatattaa?1861

gttcttatgt?atgtcaaaag?atatcatttt?gaaattcatc?catcttattg?ggtattgcag?1921

gagttcattc?ctttttgttt?ataaatactc?ttccgtcata?tgaatagtat?tcatttgtat?1981

actggtttgt?tgatggacat?ttgggttgtt?cccagtttat?ggctattaca?aataaagctt?2041

ctatgaacat?ttatgtaca 2101

3, the information of SEQ ID NO:2:

(i) sequence signature

(A) length: 95aa

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: polypeptide

(x i) sequence description: SEQ ID NO:2:

MKFLLLVLAALGFLTQVIPASAGGSKCVSNTPGYCRTCCHWGETALFMCN 50

ASRKCCISYSFLPKPDLPQLIGNHWQSRRRNTQRKDKKQQTTVTS 95

Claims (10)

1. the gene HEL-75 of people's epididymal expression is characterized in that, has the nucleotide sequence shown in the sequence table SEQ ID NO:1, and its expressing protein has the aminoacid sequence shown in the sequence table SEQ ID NO:2.

2. according to right 1 described expression of gene albumen, it is characterized in that, 3. according to right 2, it is characterized in that it being a kind of epididymis alexin, HEL-75 albumen exists a N-tetradecanoyl site (G23GSKCV) and a N-glycosylation site (N50ASR).In addition, also may there be four phosphorylation sites, comprise a cAMP-and cGMP-dependent protein kinase phosphorylation site (R79 RNT) and three protein kinase C phosphorylation sites (S52RK, S77RR and T82QR).And, in molecule, also found a potential coagulation factors V LSPD tumor-necrosis factor glycoproteins (L62PKPDLPQL).

3. according to claim 2,3 described HEL-75 albumen is characterized in that, this albumen also comprises indivedual amino acid whose disappearances, replacement or modification in the aminoacid sequence shown in the SEQ ID NO:2 and the varient or the function equivalent that obtain.

4. according to claim 1 and 2 described genetic expression albumen, belong to the sperm binding protein of people's epididymis specifically expressing, relevant with spermioteleosis and defense function.

5.HEL-75 albumen is characterized in that, can be albumen, chemosynthesis albumen, recombinant protein of natural separation and purification etc.

6.HEL-75 albumen is characterized in that described albumen can adopt the gene engineering method preparation, to contain the carrier of SEQ ID NO:1 encoding sequence, transforms protokaryon or eukaryotic cell, carries out the HEL-75 protein expression, expressing protein is through affinity chromatography method purifying.

7. according to the albumen that claim 2 obtained, it is characterized in that, can be used as corresponding mono-clonal of immunogen preparing or polyclonal antibody, be used for the proteic detection of HEL-75.Can be used for preparing protein chip, immunity detection reagent etc., the auxiliary diagnosis infertility.

8. according to claim 5, the proteic gene of coding HEL-75 can be used for developing detection methods such as new gene chip, nucleic acid probe, is used for the auxiliary diagnosis of human reproduction's medical research and clinical infertility.

9. according to claim 2 or 3 described albumen, it is a kind of pharmaceutical composition, as the effective active composition, can be mixed with one or more pharmaceutically acceptable carrier or additives with this, be used to produce a kind of medicine for the treatment of infertility or diseases such as antagonism cause pathogeny imcrobe infection and tumour.

10.HEL-75 albumen can be used as a kind of target protein of developing new contraceptive bian.

Images