CN102329378B - HCV (hepatitis C virus) core antigen and antibody thereof as well as hybridoma cell lines secreting antibody - Google Patents
HCV (hepatitis C virus) core antigen and antibody thereof as well as hybridoma cell lines secreting antibody Download PDFInfo
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Abstract
The invention discloses an HCV (hepatitis C virus) core antigen, which is a polypeptide encoded by a DNA (deoxyribonucleic acid) of which the amino acid sequence is shown in SEQ ID NO.1 and the nucleotide sequence is shown in SEQ ID NO.2. The invention also discloses a hybridoma cell line secreting monoclonal antibodies, the hybridoma cell line is named as SC16-H6 and has been preserved in the China General Microbiological Culture Collection Center on July 14th, 2011, and the preservation number is CGMCC No. 5074. The invention also discloses another hybridoma cell line secreting monoclonal antibodies, the hybridoma cell line is named as SC23-G5 and has been preserved in the China General Microbiological Culture Collection Center on July 14th, 2011, and the preservation number is CGMCC No. 5075. The invention also discloses a monoclonal antibody of an anti-HCV core antigen, which is secreted by the hybridoma cell line (preservation number: CGMCC No. 5074) or the hybridoma cell line (preservation number: CGMCC No. 5075), and has a specificity to the polypeptide shown in SEQ ID No.1.
Description
Technical field
The present invention relates to a kind of cAg, and this antigen is had specific antibody, and can secrete the hybridoma cell strain that produces this antibody to China's infection with hepatitis C virus epidemic strain.
Background technology
Hepatitis C is that (hepatitis C virus HCV) infects the transmissible disease cause, has become that another causes the viral infectious of global concern after hepatitis B, AIDS by hepatitis C virus.At present, whole world hepatitis C virus carrier has 1.7 hundred million people approximately, and with the speed increase in 300～4,000,000 people/years; China sufferer is more than 6,000 ten thousand people, and average infection rate is 3.2%, high epidemic regions in the genus, and the HCV genotype is main with 1b and 2a, wherein the 1b type accounts for more than 70%.
HCV infects and is prone to chronic conversion, and 80-85%HCV the infected develops into chronic hepatitis C, wherein 20% develops into hepatic fibrosis, and 4-5% patients with liver fibrosis generation hepatocellular carcinoma is finally arranged, and is the important factor that causes liver cirrhosis, hepatocellular carcinoma.Some data presentation infect relevant mortality ratio with HCV in following 20 years and will continue to increase.Early HCV the infected being diagnosed and giving correct treatment is the key of slowing down PD, reducing mortality ratio.Because the HCV highly complex mechanisms of variation and immunity of organism and HCV infection makes the development of hcv vaccine difficult, difficulty has breakthrough in a short time.Though Interferon, rabbit has certain curative effect to chronic hepatitis C, grow the course of treatment, cost an arm and a leg, toxic side effect is big and be prone to bounce-back.The serological anti-HCV/EIA of domestic main application at present detects antibody, because the existence of window phase behind antibody screening, still has part blood transfusion back third liver to take place clinically.Complicated, the pollution easily of the virological detecting operation of HCV costs an arm and a leg, and is difficult to extensive examination.Set up the HCV antigen early detection method of highly sensitive, specificity and stability, shorten the detection window phase, can effectively prevent to propagate, and the monoclonal antibody of acquisition high specific is a key wherein.The zone of the non-coding region at hepatitis c virus gene group two ends and coding core protein is the most conservative zone in the HCV genome.Its N holds preceding 120 amino acid to constitute main hydrophilic-structure territory, has very strong immunogenicity.This region amino acid sequence is conservative, contains a plurality of linear epitopes, about 90% homology of the amino acid of different virus strain isolated.The HCV core protein of high conservative is one of requisite antigen of HCV early diagnosis.Preparation is to the specific murine monoclonal antibody of China HCV epidemic strain, the significant and using value to setting up of core antigen of C type hepatitis virus detection method.
Adopt the monoclonal antibody of mouse-murine hybridoma technology preparation to be still one of important source of diagnostic reagent so far; Technology is comparatively ripe; Because of its high specificity; Can reduce possible cross reaction, thereby assay more accurately can be provided, the monistic advantage of homogeneity and biological activity also is beneficial to carries out stdn and normalized quality control.
Summary of the invention
To above-mentioned prior art; The invention provides a kind of cAg, and this antigen is had specific antibody, and can secrete the hybridoma cell strain (two strains) that produces this antibody to China's infection with hepatitis C virus epidemic strain; Monoclonal antibody provided by the invention; Can combine the HCV cAg with high specificity, have higher avidity, can be used for the development of population of China HCV early diagnosis cAg detection kit.
The present invention realizes through following technical scheme:
A kind of core antigen of C type hepatitis virus, its aminoacid sequence are the polypeptide of being classified as the dna encoding shown in the SEQID NO.2 by nucleotides sequence shown in SEQ ID NO.1.
The hybridoma cell strain of one strain secrete monoclonal antibody; This hybridoma cell strain is SC16-H6; Called after mouse-mouse hybridoma was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 07 14th, 2011, and deposit number is CGMCC No.5074.
The hybridoma cell strain of another strain secrete monoclonal antibody; This hybridoma cell strain is SC23-G5; Called after mouse-mouse hybridoma was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 07 14th, 2011, and deposit number is CGMCC No.5075.
Said hybridoma cell strain is through merging, screen, clone, go down to posterity and the mouse hybridoma cell system frozen repeatedly, that the recovery back obtains, ability stably excreting monoclonal antibody by the Balb/c mouse boosting cell of immunity and rat bone marrow tumour cell SP2/0.
A kind of monoclonal antibody of anti-hepatitis c virus cAg; Be to be that the hybridoma cell strain of CGMCC No.5074 or the secretion of hybridoma cell strain that deposit number is CGMCC No.5075 produce by deposit number, it has specificity to the polypeptide shown in the SEQ ID NO.1.
The said monoclonal antibody method of a kind of preparation; Comprise: with the polypeptide immune Balb/c mouse shown in the SEQ ID NO.1; Getting mouse spleen cell and myeloma cell merges; Filter out and to secrete the hybridoma that the polypeptide shown in the SEQ ID NO.1 is had specific monoclonal antibody, from hybridoma supernatant or from the animal ascites of injection hybridoma, obtain said monoclonal antibody.
Core antigen of C type hepatitis virus provided by the invention; It is the HCV core area fragment of extracting RNA and cloning through from China HCV patients serum; For 3-120 before the core area of HCV1b/2a hypotype amino acid whose albumen, contained a plurality of linear epitopes, be primarily aimed at China HCV and infect epidemic strain; Can be used for that immune mouse prepares monoclonal antibody and immunizing rabbit prepares polyclonal antibody, advantages such as antigenicity is strong, purity is high, good stability that it has.
Anti-HCV mouse monoclonal antibody can be stablized, efficiently secreted to two strain of hybridoma strains provided by the invention, has higher avidity, has important use value to setting up HCV cAg detection method.
Monoclonal antibody provided by the invention; Can be used to detect core antigen of C type hepatitis virus; Also can be used to prepare detection kit, said detection kit contains and affinity tag bonded monoclonal antibody, and said affinity tag is fluorescent marker, radioactively labelled substance or enzyme labelling thing.Concrete purposes is following:
Direct purposes: foundation can detect hepatitis C virus mutant strain and the wild strain clinical detection experimental technique at interior core antigen of C type hepatitis virus simultaneously; Comprise ELISA; The colloid gold immune dialysis; The solid phase of multiple C hepatitis virus antigen and liquid phase analysis, and panimmunity pathology detection technology etc.
Indirect purposes: the experimental technique and the reagent that adopt hybridoma cell strain secretory antibody provided by the invention to be developed can be widely used in the basis of hepatitis C, clinical and epidemiological study.
Beneficial effect of the present invention is mainly reflected in: a kind of monoclonal antibody to population of China HCV is provided; And the hybridoma cell strain that produces this monoclonal antibody; On basis of the present invention, can set up sensitivity, reliable detection method; For diagnosis, prognosis and clinical therapeutic efficacy detection theoretical basis is provided for assessing it, has had the major application prospect.
Description of drawings
Hybridoma cell strain SC16-H6, called after mouse-mouse hybridoma was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 07 14th, 2011, and deposit number is CGMCC No.5074.
Hybridoma cell strain SC23-G5, called after mouse-mouse hybridoma was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 07 14th, 2011, and deposit number is CGMCC No.5075.
Fig. 1 is an antigen restriction enzyme digestion and electrophoresis synoptic diagram.
Fig. 2 identifies synoptic diagram for protein antigenicity western.
Fig. 3 A is a purifying protein electrophoresis synoptic diagram.
Fig. 3 B identifies synoptic diagram for purifying protein western.
Fig. 4 analyzes synoptic diagram for antigenic index.
Fig. 5 is the chromosome analysis synoptic diagram.
Fig. 6 is a monoclonal antibody hypotype electrophoresis synoptic diagram.
Fig. 7 identifies synoptic diagram for the monoclonal antibody specificity.
Embodiment
Below in conjunction with accompanying drawing invention is further described.
Embodiment one immunogenic preparation
1. design and synthesize HCV core gene fragment primer sequence (synthetic), like SEQ ID NO.3, shown in 4 by Shanghai Bo Shang Bioisystech Co., Ltd.
2. extract the HCV infected patient and (be used for the HCV-RNA positive that 20 routine HCV serum samples that HCV RNA extracts collect from Shandong Qilu Hospital but HCV negative antibody patient; The male sex's 16 examples wherein, women's 4 examples, 45 years old mean age; Through the genotype tests result is 13 routine Ib hypotypes; 7 routine 2a hypotypes) serum RNA carries out reverse transcription PCR, and reaction system is following:
Carry out the PCR reaction by following condition: 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30s, after 35 circulations, 72 ℃ of 10min.
3. glue recovery purpose PCR product is connected with the pMD18-T carrier behind the agarose gel electrophoresis, is transformed in the DH5 α engineering bacteria.Picking list bacterium colony shakes bacterium extracting plasmid, cuts evaluation through enzyme, to confirm obtaining the evaluation of checking order of the segmental engineering bacteria of purpose.
4. recombinant plasmid and PET-30a are used NdeI and HindIII double digestion respectively; Glue reclaims the purpose fragment behind the agarose gel electrophoresis, connects through the T4 ligase enzyme, is transformed into the BL21-DE3 competence; Picking list bacterium colony shakes behind the bacterium with PCR method screening positive clone; Amplified production is identified (as shown in Figure 1, on behalf of 5 strain bacterium, M, 1,2,3,4,5 represent marker) with agarose gel electrophoresis, send order-checking.
5. the efficient induction of positive colony is expressed
(1) the abduction delivering screening efficiently expresses the clone and identifies the antigenic antigenicity of HCV in a small amount
Behind 37 ℃ of activation 12h of bacterium with sequencing result correct (sequencing result is shown in the SEQ ID NO2, is correct), by 1: 100 concentration inoculation medium, 37 ℃ were shaken bacterium 3h, added isopropylthiogalactoside (IPTG) and induced 4h.Respectively getting bacterium liquid after inducing concentrates and to add sample solution and do the PAGE electrophoresis, coomassie brilliant blue staining visual inspection purpose band, the clone that preliminary screening goes out to efficiently express.Make Western blot with SDS-PAGE, His tag antibody and HCV polyclonal antibody; Identify engineered protein antigenicity (Fig. 2), can find out that this bacterium induces the expressed albumen in back to contain the His label by Fig. 2; But the application of nickel post is further purified, and HCV is had avidity preferably.
(2) optimize inductive condition
Make the gradient abduction delivering with differing temps, different induction time and different IP TG concentration; Temperature is respectively 20 ℃, 25 ℃, 30 ℃, 37 ℃; Induction time is 1h, 2h, 4h, 6h, 8h, 24h, and IPTG concentration is respectively 0.1,0.3,0.5,1.0,3.0mmol/L.Collect inductive bacterium liquid detection expressing quantity under the different condition, and the clear and definite albumen existence form in ultrasonic back.Best inductive condition: 37 ℃, IPTG concentration are that 1.0mmol/L induces 4h.It is inclusion body that there is principal mode in albumen.
(3) great expression of recombinant protein and purifying
Induce a large amount of bacterium liquid with above-mentioned condition, 4 ℃ of centrifugal collection thalline of 8000rpm, PBS are washed back 20 times and are concentrated;-40 ℃ are spent the night, and carry out ultrasonication in the ice bath, the centrifugal collection albumen of 12000rpm inclusion body; By 10: 1 adding lysates; 37 ℃ of 3h, with saturated urea dissolving and further through ni-sepharose purification, eluted product is removed urea through dialysis.The BCA method is measured protein concentration.
(4) the antigenic evaluation of HCV core protein
Purifying protein SDS-PAGE and Western Blot see Fig. 3, and Fig. 4 is seen in the antigenic index analysis.The SDS-PAGE method is identified the antigen purity behind the ni-sepharose purification, and the protein applied sample amount is 10 μ l, can be found out by Fig. 3 A, only deposits a band, and purity is higher.Make Western blot with the HCV polyclonal antibody, Fig. 3 B shows that the albumen behind the purifying has antigenicity preferably.Utilize DNAstar-protean software to carry out the antigenic index analysis, as shown in Figure 4, this proteic epitope concentrates on 1-25,35-42,46-73 and 96-118 amino acid.
Embodiment two hybridomas prepare monoclonal antibody
1. animal immune:
Choose the female mouse of Balb/c in 6 ages in week, subcutaneous multi-point injection immunogen is used Freund's complete adjuvant, mixes by 1: 1, and initial immunity used the incomplete Freund's adjuvant subcutaneous injection after 10 days, and is weekly, continuous three times.After the last immunity, blood is got in docking, tires through indirect ELISA method preliminary evaluation mouse antibodies.After 10 days, booster immunization once.
2. cytogamy:
Before the fusion, recovery rat bone marrow tumour cell SP2/0, and carry out screening and culturing with the 8-azaguanine, cell is in the optimum growh state when guaranteeing to merge.Merge the same day, get the mouse peritoneal macrophage and prepare feeder cell, add 10ml HAT nutrient solution branch and plant in 96 orifice plates.
Behind the immune mouse booster immunization three days, get its splenocyte and rat bone marrow tumour cell by 5-10: 1 mixes, centrifugal after, exhaust supernatant as far as possible, touch the pipe end, make sedimentation cell loose evenly, dropwise add 40% PEG40000.7ml, the limit edged stirs gently.Dropwise slowly add serum-free 1640 substratum subsequently.Added 0.7ml in first minute, fluid infusion subsequently is to 10ml, and centrifugal 5 minutes of 800rpm abandons supernatant.Slowly add the HAT substratum, pressure-vaccum sedimentation cell gently, back fluid infusion divides kind in 96 orifice plates to 10ml, 37 ℃ of 5%CO
2Cultivate in the incubator.Merge after 3 days, the HAT substratum of HT substratum replacement 1/2 merges after 5-7 days wholly replace HAT substratum.Through observing, cell confluency can reach more than 80%.Close observation cell growing state is when cell grows to 1/2 or during the above visual field, the sucking-off supernatant detects antibody with indirect ELISA method.
3. hybridoma screening:
Chessboard method confirms that best immunizing antigen encapsulates concentration and enzyme labelled antibody working concentration, adopts indirect ELISA method to carry out filtering hybridoma.Positive control is the immune mouse polyvalent antibody, and negative control is mice serum before the immunity, and is positive with ratio＞2.1 of control value with measured value.
4. hybridoma subcloning:
Adopt limiting dilution assay carry out cell cloneization to positive colony.Cloning same day, get Turnover of Mouse Peritoneal Macrophages and divide kind in 96 orifice plates.Cell counting adjustment cell density is carried out in the positive colony hole, by cell number be 2,1,0.5 extent of dilution kind in 96 orifice plates, by each extent of dilution 32 hole, every hole 100ul.At 37 ℃, cultivated about 7 days in the 5%CO2 incubator, ELISA carries out antibody titer and detects.Select the high mono-clonal hole of antibody titer, repeated cloningization 3 times detects cloning hole 100% positive in definite final 96 orifice plates through ELISA.Set up the cell strain of stably excreting antibody, and carry out preservation, be respectively SC23-G5 (CGMCC No.5075) and SC16-H6 (CGMCC No.5074).
5. Monoclonal Antibody
Change the positive porocyte of mono-clonal over to 24 holes earlier, change over to subsequently and carry out enlarged culturing in the culturing bottle.The 0.5ml whiteruss is injected the Balb/c mouse peritoneal.Every mouse peritoneal inoculation 0.2ml (about 5 * 10 after 7 days
5) individual cell.Observe mouse web portion every day after 5 days, when skin has the anxiety sense when touching with hand, promptly gathers ascites with the 16-18 syringe needle, extracts once every other day, whenever adopts 5-10ml ascites only.Centrifugal cell and other throw out abandoned collected supernatant, and mensuration is tired, and anti-body contg is measured and is respectively 6.5mg/ml, 6.0mgml.Branch is installed on-70 ℃ and deposits.
6. the evaluation of monoclonal anti bulk properties
(1) chromosome analysis of hybridoma: adopt the NST-757 method to analyze the karyomit(e) of two strain of hybridoma.The selective staining body is scattered during microscopy, and zero lap does not have the cell that scatters and carries out observation analysis (Fig. 5).
Detailed process does, before adding NST-757, hybridoma gone down to posterity in 48-36 hour, and in culturing bottle, adding final concentration is the NST-757 of 0.1ug/ml, continues to cultivate 6 hours, with the negative contrast of logarithmic phase SP2/0 cell.Blow and beat cell gently with suction pipe, move in the centrifuge tube, centrifugal 10 minutes of 1000r/min abandons supernatant; Add and to be preheating to 37 ℃ 0.075mol/LKCl solution 5ml, 37 ℃ of water-baths 15 minutes; In suspension, add stationary liquid (methyl alcohol mixes with Glacial acetic acid min. 99.5 at 3: the 1) 1ml of new preparation, mixing left standstill 2 minutes, and centrifugal 10 minutes of 1000r/min abandons supernatant; Add stationary liquid 5ml, featheriness cell suspension and mixing, room temperature left standstill 20 minutes, the centrifugal supernatant of abandoning; Repetitive operation once; Add the 5ml stationary liquid then, mixing, 4 ℃ are spent the night; Take out centrifuge tube, 1000r/min 5 minutes inhales gently and removes supernatant, adds the 0.5ml stationary liquid, and behind the piping and druming mixing, 1-2 drips to the unsettled dropping cell suspension of the slide glass of precooling, and it is fixing on spirit lamp flame to dispel the back.10%Giemsa dye liquor with new preparation dyeed 20 minutes, used tap water flush away dye liquor then, seasoning; Mirror is observed down.
(2) monoclonal antibody subgroup identification and titration: adopt the ELISA method that two strain of hybridoma excretory antibody are carried out subgroup identification.Detailed process does, PBS dilution hypotype antibody, every hole adds 100ul, 4 ℃ spend the night after, the PBST washings is washed 3 times; Every hole adds the 200ul confining liquid, room temperature effect 1 hour, and washings is washed 3 times; Add 100ul Hybridoma Cell Culture supernatant, room temperature effect 1 hour, PBST washes 3 times; Every hole adds 100ul Ig subclass ELIAS secondary antibody, room temperature 1 hour, and PBST washes 5 times; Add the substrate colour developing, sentence read result.Two strain monoclonal antibodies are Er Tiao district band (see figure 6) on SDS-PAGE.
With normal SP2/0 cell and the negative contrast of corresponding ascites, with the cells and supernatant of serial dilution with induce ascites and add the enzyme plate that the HCV cAg encapsulates, hatched 1 hour for 37 ℃ in 100 μ l/ holes; After washing plate, add the HRP mark sheep anti-mouse igg of dilution in 1: 1000, hatched 1 hour for 37 ℃ in 100 μ l/ holes; Add colour developing liquid colour developing back termination reaction, ELIASA is measured the OD450nm value, and measured value is judged to the positive with ratio＞2.1 of negative control value, tires as monoclonal antibody with the greatest dilution in positive hole.The result shows that monoclonal antibody is tired in SC23-G5 and the SC16-H6 cells and supernatant and all is about 1: 100 that tiring of monoclonal antibody is respectively 1: 6400 and 1: 12800 in the ascites.
(3) the specific evaluation of monoclonal antibody: the monoclonal antibody that obtains is identified with Western Blot method; HCV cAg, NS3 proteantigen and tropina are carried out the SDS-PAGE electrophoresis, and electrotransfer seals after 2 hours to nitrocellulose membrane; Add the cells and supernatant that contains monoclonal antibody; Wash the sheep anti-mouse igg that adds the HRP mark behind the film, oscillatory reaction 1 hour adds the substrate colour developing after washing film.PRELIMINARY RESULTS shows that two strain monoclonal antibodies all only react to the HCV cAg, and the equal Fails To Respond of tropina to other proteantigen and expression cAg proves to have specificity (Fig. 7) preferably.
Claims (7)
1. core antigen of C type hepatitis virus, it is characterized in that: its aminoacid sequence is a polypeptide of being classified as the dna encoding shown in the SEQ ID NO.2 by nucleotides sequence shown in SEQ ID NO.1.
2. the hybridoma cell strain of a strain secrete monoclonal antibody; It is characterized in that: this hybridoma cell strain called after SC16-H6; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 07 14th, 2011; Deposit number is CGMCC No.5074, and it is the monoclonal antibody to cAg shown in the aminoacid sequence SEQ ID NO.1 of claim 1.
3. the hybridoma cell strain of a strain secrete monoclonal antibody; It is characterized in that: this hybridoma cell strain called after SC23-G5; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 07 14th, 2011; Deposit number is CGMCC No.5075, and it is the monoclonal antibody to cAg shown in the aminoacid sequence SEQ ID NO.1 of claim 1.
4. the monoclonal antibody of an anti-hepatitis c virus cAg; It is characterized in that: said monoclonal antibody is to be that the hybridoma cell strain secretion of CGMCC No.5074 produces by deposit number, and it has specificity to the polypeptide shown in the aminoacid sequence SEQ ID NO.1 of claim 1.
5. the application of the described monoclonal antibody of claim 4 in the preparation detection kit, it is characterized in that: said detection kit contains and affinity tag bonded monoclonal antibody, and said affinity tag is fluorescent marker, radioactively labelled substance or enzyme labelling thing.
6. the monoclonal antibody of an anti-hepatitis c virus cAg; It is characterized in that: said monoclonal antibody is to be that the hybridoma cell strain secretion of CGMCC No.5075 produces by deposit number, and it has specificity to the polypeptide shown in the aminoacid sequence SEQ ID NO.1 of claim 1.
7. the application of the described monoclonal antibody of claim 6 in the preparation detection kit, it is characterized in that: said detection kit contains and affinity tag bonded monoclonal antibody, and said affinity tag is fluorescent marker, radioactively labelled substance or enzyme labelling thing.
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| CN109738648B (en) * | 2018-12-29 | 2021-09-17 | 山东莱博生物科技有限公司 | Engineering cell strain for stably and efficiently expressing hepatitis C virus core antigen antibody and application thereof |
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| CN102718840B (en) * | 2012-08-06 | 2014-05-14 | 中山大学 | Human MMP-14 antigen, corresponding monoclonal antibody and application thereof |
| CN109678955B (en) * | 2018-12-29 | 2022-01-07 | 山东莱博生物科技有限公司 | Monoclonal antibody pair for detecting HCV-cAg, hybridoma cell strain secreting antibody pair and application of monoclonal antibody pair |
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| CN1105703A (en) * | 1994-08-25 | 1995-07-26 | 中国药品生物制品检定所 | Building of anti-type-C hepatitis virus antigen monoclonal antibody cell strain and monoclonal antibody thereof |
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