CN103276009A - Construction and expression for campylobacter jejuni cytolethaldistending toxin expression vectors, and preparation for monoclonal antibody - Google Patents
Construction and expression for campylobacter jejuni cytolethaldistending toxin expression vectors, and preparation for monoclonal antibody Download PDFInfo
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Abstract
The invention relates to construction and expression for campylobacter jejuni cytolethaldistending toxin expression vectors, and preparation for a monoclonal antibody. The campylobacter jejuni cytolethaldistending toxin expression vectors are pET-cdtC and pGEX-6p-1-cdtC. A preparation method for an anti-CdtC monoclonal antibody comprises the following steps of: (1) performing prokaryotic expression and purification on the proteins of his-CdtC and GST-CdtC; (2) immunizing BABL/c mice by GST-CdtC, fusing the splenocytes of the mice with high titers with SP2/0 cells, and culturing and screening cell strains secreting a specific antibody; (3) filling the screened cell strains in the abdominal cavities of the BABL/c mice, so as to obtain high-concentration monoclonal antibody, namely, to finish preparation for the monoclonal antibody; and (4) determining the titers of the supernatant and the ascites of the monoclonal antibody.
Description
Technical field
The present invention relates to two kinds of expression vectors, particularly two kinds of expression vectors of cell lethality swelling toxin cdtC gene.The invention still further relates to structure, expression and the MONOCLONAL ANTIBODIES SPECIFIC FOR of this expression vector.
Background technology
Campylobacter jejuni is a kind of important former bacterium of food source property Amphixenosis, is to cause in the global range that the human gastrointestinal tract infects modal pathogenic bacteria.In recent years, in rising trend by the microbial human diseases of jejunum campylobacter, its Study on Pathogenicity has been become a hot issue.
Its pathogenesis research is mainly concentrated on outer membrane protein, structural protein and toxin, cell lethality swelling poison (Cytolethal distendin toxin, CDT) be a kind of albumen that is produced by gram-negative bacteria, it has cytotoxicity, can name a person for a particular job the cell cycle blocking-up in the G2/M phase by damage dna and activating cells cyclic check, cause cellular swelling, death.Cell lethality swelling toxin has been considered to the important pathogenic factors that campylobacter jejuni infects.
Genes involved cdtA, the cdtB that the cell lethality swelling toxin of campylobacter jejuni is arranged by three kinds of karyomit(e)s, peptide C dtA, CdtB and the CdtC that the operon of cdtC is encoded into constitute.CdtB is the key component of cell lethality swelling toxin, is the active subunit of this toxin.CdtA is responsible for being bundled in cell surface, and CdtC helps CdtB to show the toxin activity.The CdtC of campylobacter jejuni is essential or can strengthens cytotoxic activity at least, and this subunit may be from inducing human epithelium INT407 clone at release in vitro interleukin-(IL)-8, and the CdtC specific antibody can suppress toxicity.This kind toxin is a kind of extracellular toxin, directly directly extracts relatively difficulty of this kind albumen from the culture supernatant of bacterium.
Summary of the invention
The object of the present invention is to provide two kinds of recombinant plasmid vectors, this carrier all contains the cdtC gene.
Another object of the present invention is to above-mentioned carrier proteins in the application of the monoclonal antibody of the anti-cdtC of preparation.
The present invention makes up and to contain cdtC Prokaryotic Expression plasmid, the expression in escherichia coli Histidine (Histidine, His)-CdtC fusion rotein glutathione sulfydryl transferase (Glutathione S-transferase, GST)-the CdtC fusion rotein.For getting rid of the influence of label, be immunogen with GST-CdtC, His-CdtC is former for detecting.For the monoclonal antibody for preparing CdtC lays the foundation.
Anti-CdtC MONOCLONAL ANTIBODIES SPECIFIC FOR method realizes according to the following steps: one, prokaryotic expression and the purifying of His-CdtC, GST-CdtC albumen; Two, with GST-CdtC immunity BABL/c mouse, get splenocyte and the SP2/0 cytogamy of the higher mouse of potency ratio, cultivate and filter out the cell strain of secreting specificity antibody; Three, the cell strain with screening injects the BABL/c mouse peritoneal, obtains the monoclonal antibody of high density, namely finishes MONOCLONAL ANTIBODIES SPECIFIC FOR; Four, to the supernatant of monoclonal antibody, the ascites titration, subclass is measured and the specificity experiment.
Wherein step 1 adopts pET-30a, the pGEX-6p-1 prokaryotic expression carrier, and 2 kinds of carriers contain different labels, and the pET-30a carrier contains the His label, and pGEX-6p-1 contains the GST label.The recombinant plasmid dna measurement result has confirmed to contain goal gene.To recombinate bacterium BL21 (DE3) (pET-cdtC) and BL21 (pGEX-6p-1-cdtC) be inoculated in respectively to contain in Ka Na and the antibiotic substratum of ammonia benzyl and cultivate the IPTG abduction delivering.Centrifugal collection thalline, SDS-PAGE analyzes.Expression product is the syzygy of His and destination gene expression albumen when genetic expression, the syzygy of GST and destination gene expression albumen.2 kinds of fusion roteins size be 36KD, 47KD.
Step 3 is to the 9-11 female BALB/c mouse abdominal injection whiteruss in age in week (0.3-0.5ml/ only), and behind the 7-10d, the hybridoma that will be in logarithmic phase suspends (5 * 10 with PBS
5Individual/0.3mL), abdominal injection.Treat about 7 days that mouse web portion obviously expands, gather ascites, centrifugal collection supernatant ,-70 ℃ of preservations.
Step 4 is utilized ELISA method mensuration to go up cleer and peaceful ascites and is tired, and identifies the antibody subclass by monoclonal antibody subclass test kit, the specificity of Western-blot test evaluation monoclonal antibody.
Description of drawings
Fig. 1 is cdtC gene PCR amplification figure; M:DL2000; 2:Amplification of cdtC gene.
Fig. 2 is that recombinant plasmid pMD-18-T-cdtC enzyme is cut the evaluation collection of illustrative plates; M:DL2000,1:pMD-18T-cdtC/BamH I+Xho I.
Fig. 3 is the cleavage map of recombinant plasmid pET-cdtC, pGEX-6p-1-cdtC; 1:pET-32a-cdtC/BamH I+Xho I; 2:pGEX-6p-1-cdtC/BamH I+Xho I.
Fig. 4 recombinates bacterium BL21 (DE3) (pET-cdtC), the SDS-PAGE of BL21 (pGEX-6p-1-cdtC);
1:BL21(DE3)(pET-cdtC)induced?by?IPTG
2:Supernatants?of?lysate?of?BL21(DE3)(pET-cdtC)induced?by?IPTG
3:BL21(DE3)(pET)induced?by?IPTG
4:BL21(pGEX-6p-1-cdtC)induced?by?IPTG
5:Supernatants?of?lysate?of?BL21(pGEX-6p-1-cdtC)induced?by?IPTG
6:BL21(pGEX-6p-1)induced?by?IPTG。
Fig. 5 is the His-CdtC of purifying, GST-CdtC protein;
1:purified?GST-CdtC?protein
2:purified?His-CdtC?protein。
Fig. 6 is that the Western-blot of 5 strain CdtC monoclonal antibodies analyzes
M:protein?marker
3:BL21(pGEX-6p-1-cdtC)whole?protein
4:BL21(pGEX-6p-1)whole?protein
5:BL21(DE3)(pET-cdtC)whole?protein
6:BL21(DE3)(pET)whole?protein。
Embodiment
The amplification of embodiment 1, cdtC gene
The sequence that goes up the cdtC gene of announcing according to GenBank designs primer respectively, and the primer of design does not contain signal peptide, and primer sequence is as follows:
CdtC:F(contains BamH I restriction enzyme site): TAG
GATCCTGGATGATAGCAGGGGATTTTAAC(SEQ ID NO.1)
R(contains the XhoI restriction enzyme site): ATCTCGA
GTCTTATTCTAAAGGGGTAGC(SEQ ID NO.2)
Be template with campylobacter jejuni type strain NCTC11168 genomic dna, amplification system is: the total reaction system of PCR is 50 μ l:
The PCR loop parameter is: 94 ℃ of pre-sex change 10min; 94 ℃ of sex change 1min, 60 ℃ of annealing 1min, 72 ℃ are extended 1min, 35 circulations, 72 ℃ are extended 10min.
Get PCR product 5ul, identify with 1.2% agarose gel electrophoresis, and reclaim its product.
The product that the PCR of cdtC gene is reclaimed is connected with cloning vector, recombinant plasmid after the connection is through BamH I and the two enzymes of Xho I, obtain 699bp and 2.7kb respectively, the fragment of 519bp and 2.7kb size, meet with the expection size, sequencing result shows that also the cdtC gene that experiment obtains and the cdtC gene order of NCTC11168 type strain are in full accord.
Structure and the evaluation of embodiment 3, reorganization prokaryotic expression plasmid pET-cdtC, pGEX-6p-1-cdtC
Reclaim test kit purifying purpose fragment cdtC with glue, the pGEX-6p-1 that cuts with enzyme, the pET expression vector connects, change DH5 α competence bacterium over to, through blue hickie screening, enlarged culturing, the upgrading grain, enzyme is cut evaluation, changes plasmid pET-cdtC, pGEX-6p-1-cdtC over to BL21 and BL21 (DE3) competence bacterium after evaluation is correct, the recombinant plasmid transformed of pET is to BL21 (DE3) competence bacterium, the recombinant plasmid transformed of pGEX-6p-1 is to BL21 competence bacterium, picking list bacterium colony, enlarged culturing, the upgrading grain, enzyme is cut evaluation.Recombinant expression vector pET-cdtC is behind BamH I and Xho I double digestion, and two bands about 5.9kb and 519bp appear in electrophoresis.Recombinant expression vector pGEX-6p-1-cdtC is behind BamH I and Xho I double digestion, and two bands about 4.9kb and 519bp appear in electrophoresis.More than all with the expection size meet.Show that goal gene successfully inserts in the expression vector.
Embodiment 4, the expression of cdtC gene in the reorganization bacterium
The recombination bacillus coli BL21 (DE3) of fresh culture (pET-cdtC) and BL21 (pGEX-6p-1-cdtC) be inoculated in 1:100 and contain in the antibiotic liquid LB substratum, 37 ℃ of shaking culture, when being cultured to the OD value and being about 0.4 left and right sides, adding final concentration is the IPTG of 0.5mM/L, induce 4h for 37 ℃, centrifugal collection thalline, PBS washing, suspension.Utilize ultrasonic disruption instrument cracking thalline in ice bath.SDS-PAGE identifies.Get reorganization bacterium BL21 (DE3) respectively (pET-cdtC) as figure, whole bacterial protein after BL21 (pGEX-6p-1-cdtC) induces, cracking supernatant, cracking precipitation are carried out the SDS-PAGE electrophoresis, simultaneously with empty carrier bacterium BL21 (DE3) (pET), BL21 (pGEX-6p-1) in contrast.The SDS-PAGE electrophoresis result shows: (pET) compare with the empty carrier bacterium BL21 (DE3) of swimming lane 3, the reorganization bacterium BL21 (DE3) of swimming lane 1 is that specific band appears in the 35KD place at molecular weight (pET-cdtC), and the achieving success expression in the reorganization bacterium of cdtC gene is described; There is the purpose band in swimming lane 1 cracking precipitation, and swimming lane 2 also has the purpose band for the cracking supernatant.The expression product that the reorganization bacterium is described is that solubility and inclusion body form exist.Compare with the empty carrier bacterium BL21 (pGEX-6p-1) of swimming lane 6, the reorganization bacterium BL21 (pGEX-6p-1-cdtC) of swimming lane 4 is that specific band appears in the 44KD place at molecular weight, and the achieving success expression in the reorganization bacterium of cdtC gene is described; Swimming lane 4 cracking precipitation have the purpose band, and swimming lane 5 does not have the purpose band for the cracking supernatant.The expression product that the reorganization bacterium is described is that the inclusion body form exists.
Embodiment 5, protein purification
The bacterium BL21 (DE3) that will recombinate (pET-cdtC) is inoculated in 1:100 and contains enlarged culturing in the antibiotic LB substratum, and the bacterium after inducing is centrifugal, collects thalline.With PBS washing and suspension, under condition of ice bath, utilize ultrasonic disruption instrument (the ultrasonic 10s of 800w, 20s at interval) cracking thalline.6000rpm10min, 8000rpm10min, 9000rpm20min successively, the centrifugal cracking supernatant of 10000rpm20min.Supernatant is collected in centrifugal back, adds among the affinity column His-Bind purification kit (washing with 20mlPBS in advance).Add 20ml binding buffer then successively, 2ml washing buffer, 12ml elute buffer collects product at last.The acquisition protein concentration is 0.8mg/ml.
BL21 (pGEX-6p-1-cdtC) is inoculated in 1:100 contains enlarged culturing in the antibiotic LB substratum.To induce the thalline of collection, every gram thalline is resuspended with 10ml buffer A, under condition of ice bath, utilize the abundant cracking thalline of ultrasonic disruption instrument after, 4 ℃ of 10000rpm15min collecting precipitations.Use buffer A resuspended again, repeat above process 3 times.The precipitation of collecting is dissolved in the sex change damping fluid, places 37 ℃ of shaking tables, 250rpm 4h, centrifugal 12000rpm 25min collects supernatant.Renaturation buffer buffer B slowly is added drop-wise to metaprotein solution (also continuous mixing under condition of ice bath), places 4 ℃, behind the 24h.With renaturation buffer buffer C(1:10) dialyse 3 times.At last protein solution is concentrated 10000rpm15min with PEG.Collect supernatant, supernatant is the albumen of purifying.Buffer A (20mmol/LTris-HCl, 50mmol/L NaCl, 1mmol/L EDTA, 1mol/L urea, 0.5% Triton-100, pH 8.0), sex change damping fluid (6mol/L Guanidinium hydrochloride, 50mmol/L Tris-HCl, 5mmol/LEDTA, 50mmol/L DTT, pH8.5), renaturation buffer buffer B (50mmol/L Tris-HCl, 1.5mol/L urea, 50mmol/L NaCl, 1mmol/L EDTA, 5% glycerine, pH8.0), renaturation buffer buffer C (20mmol/L Tris-HCl, 50mmol/L NaCl, 1mmol/L EDTA, 5% glycerine, pH8.0), obtaining the GST-CdtC protein concentration at last is 2.4mg/ml.
Embodiment 6, anti-CdtC MONOCLONAL ANTIBODIES SPECIFIC FOR
(1) immune animal
Choose the 6-8 female BALB/c mouse in age in week.First immunisation: the GST-CdtC albumen (100ug/ only) that chapter 1 is obtained mixes with isopyknic Freund's complete adjuvant, and fully emulsified, subcutaneous multiple spot immunity.Immunity again: first immunisation is after 2 weeks, and GST-CdtC albumen (100ug/ only) mixes with isopyknic Freund's incomplete adjuvant, subcutaneous injection after fully emulsified.The blood sampling of 7d posterior orbit is measured and is tired.Merging first three day, the GST-CdtC albumen of tail vein injection purifying.
(2) cytogamy
Merge one day sp2/0 and change the liquid cultivation, gently sp2/0 is blown down from the cell bottle, counting adds fusion pipe with sp2/0, immunized mice is plucked eyeball get blood, preserves separation of serum for-20 ℃, as positive control.Mouse is put to death in the cervical vertebra dislocation, 75% alcohol disinfecting 5min, and extracting spleen cell under aseptic condition adds fusion pipe, mixing, the centrifugal supernatant of abandoning.At PEG(MW4000) following these the 2 kinds of cells of fusion of effect.Get the ICR Turnover of Mouse Peritoneal Macrophages under the aseptic condition as feeder cell.As substratum, mixing and bed board place 37 ℃, 5%CO with HAT
2Cultivate in the incubator.Add fresh HAT substratum behind the 5d, change the HT culture medium culturing behind the 10d into, observe and detect.
(3) screening of positive cell clone and cloning
Be to detect formerly with His-CdtC, the Hybridoma Cell Culture supernatant added in advance bag by in the good elisa plate, the 100ul/ hole, simultaneously with the serum of immunized mice as positive control, with the SP2/0 cells and supernatant as negative control.37 ℃ of 2h, PBST washing 3 times, the sheep anti-mouse igg antibody of adding HRP mark, 100 μ l/ holes, 37 ℃ of 1h, washing is colour developing also, stops back mensuration OD
492P/N value 〉=2.1 are judged to be the positive.Utilize limiting dilution assay that positive hybridoma cell is carried out subclone 2 times.
(4) preparation of ascites
The female BALB/c mouse abdominal injection whiteruss in age in 9-11 week (0.3-0.5ml/ only), behind the 7-10d, the hybridoma that will be in logarithmic phase suspends (5 * 10 with PBS
5Individual/0.3mL), abdominal injection.Treat about 7 days that mouse web portion obviously expands, gather ascites, centrifugal collection supernatant ,-70 ℃ of preservations.
(5) evaluation of monoclonal antibody
Adopting indirect ELISA method mensuration to go up cleer and peaceful ascites tires.Utilize kit measurement monoclonal antibody subclass.The specificity of monoclonal antibody is identified in the Western-blot test.5 strain CdtC monoclonal antibodies all can with BL21 (DE3) (pET-cdtC), the protein-specific of BL21 (pGEX-6p-1-cdtC) abduction delivering is combined, and respectively at 37KD, occurs specific band about 46KD, do not have cross reaction substantially with empty carrier.Present embodiment has obtained the monoclonal antibody of specific CdtC.
Claims (3)
1. campylobacter jejuni cell lethality swelling toxin expression vector is pET-cdtC and pGEX-6p-1-cdtC.
2. anti-CdtC MONOCLONAL ANTIBODIES SPECIFIC FOR method may further comprise the steps:
(1) prokaryotic expression and the purifying of His-CdtC, GST-CdtC albumen;
(2) with GST-CdtC immunity BABL/c mouse, get splenocyte and the SP2/0 cytogamy of the higher mouse of potency ratio, cultivate and filter out the cell strain of secreting specificity antibody;
(3) with the cell strain immunity BABL/c mouse of screening, obtain the monoclonal antibody of high density, namely finish MONOCLONAL ANTIBODIES SPECIFIC FOR.
3. method as claimed in claim 2 is characterized in that, wherein step (1) adopts pET-30a, the pGEX-6p-1 prokaryotic expression carrier, and 2 kinds of carriers contain different labels, and the pET-30a carrier contains the His label, and pGEX-6p-1 contains the GST label.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399344A (en) * | 2016-06-01 | 2017-02-15 | 上海领潮生物科技有限公司 | Expression of vin-cdtb in escherichia coli and preparation method of vin-cdtb |
| CN111793130A (en) * | 2019-03-20 | 2020-10-20 | 华中农业大学 | Haemophilus parasuis CdtB hybridoma cell and application of monoclonal antibody |
| CN113462654A (en) * | 2021-08-16 | 2021-10-01 | 扬州大学 | Helicobacter hepaticus CdtB protein monoclonal antibody, hybridoma cell strain secreted by same and application of monoclonal antibody |
-
2013
- 2013-05-24 CN CN2013101985735A patent/CN103276009A/en active Pending
Non-Patent Citations (3)
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| BRUCE J.ET AL.,: "Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin (Cdt): Evidence That the Holotoxin Is Composed of Three Subunits: CdtA, CdtB, and CdtC", 《THE JOURNAL OF IMMUNOLOGY》 * |
| 刘卫卫 等: "空肠弯曲菌cdtA,cdtB及cdtC真核表达重组载体的构建与表达", 《脑与神经疾病杂志》 * |
| 刘卫卫: "空肠弯曲菌cdtA、cdtB、cdtC基因构建与表达", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399344A (en) * | 2016-06-01 | 2017-02-15 | 上海领潮生物科技有限公司 | Expression of vin-cdtb in escherichia coli and preparation method of vin-cdtb |
| CN106399344B (en) * | 2016-06-01 | 2021-01-01 | 上海领潮生物新材料有限公司 | Preparation method of vin-cdtb fusion protein |
| CN111793130A (en) * | 2019-03-20 | 2020-10-20 | 华中农业大学 | Haemophilus parasuis CdtB hybridoma cell and application of monoclonal antibody |
| CN113462654A (en) * | 2021-08-16 | 2021-10-01 | 扬州大学 | Helicobacter hepaticus CdtB protein monoclonal antibody, hybridoma cell strain secreted by same and application of monoclonal antibody |
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Application publication date: 20130904 |