CN100410274C - Specific anti-human liver cancer phage single-chain antibody HscFv4-16 gene and its uses - Google Patents
Specific anti-human liver cancer phage single-chain antibody HscFv4-16 gene and its uses Download PDFInfo
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Abstract
The present invention relates to the technical field of a genetic engineering antibody, particularly to a specific gene of a single-chain antibody HscFv4-16 of a human liver cancer phage and an application thereof. In the present invention, one specific gene scFv of an anti-human liver cancer phage, namely HscFv4-16, is selected from a built single-chain antibody library for anti-human liver cancer phage, the expressed anti-human liver cancer scFv has high specific effects on the liver cancer cell line and the human liver cancer tissue, the minimum combination titer is 256 times lower than that of the fetal liver cell line of the normal human, the stomach cancer cell line and the colon cancer cell line, the positive reaction rate with the human liver cancer tissue is 35.5%, the positive reaction rate with the liver cirrhosis tissue and the chronic hepatitis tissue is 6.7%, and the positive reaction rate with the stomach cancer tissue and the colon cancer tissue is 5.0%. The gene HscFv4-16 does not take a combination reaction with the adult normal liver tissue, the oesophagus cancer tissue and the lung cancer tissue, and the positive rate of the liver cancer tissue is obviously higher than that of the other tissues. The antibody HscFv4-16 has strong specificity to liver cancer, and can be developed into various medicines and relevant reagents for treating liver cancer.
Description
Technical field
The present invention relates to the genetic engineering antibody field, be specifically related to a kind of specific anti-human liver cancer phage single-chain antibody (Single chain fragment variable, scFv) HscFv 4-16 gene and application thereof.
Background technology
Liver cancer is one of malignant tumour of current serious harm human health, and people are seeking the various approach of diagnosis and treatment liver cancer always for many years.Continuous development along with tumor immunology and Protocols in Molecular Biology, the method that obtains anti-liver cancer scFv at present has two kinds, the one, utilize genetic engineering technique that liver cancer-resisting monoclone antibody is transformed into scFv, the anti-liver cancer scFv that has been transformed into has: Hep27 scFv, scFv25, HAb18 scFv and MDscFv, these scFv have identical specificity with their parental antibody, its antigen binding site does not change, and using value is subjected to the restriction of parental antibody.The 2nd, utilize display technique of bacteriophage to make up anti-liver cancer phage scFv storehouse, obtain new anti-liver cancer scFv by screening.But, about the screening of anti-liver cancer scFv with identify that the report of further investigation is less, so far, rarely seenly carry out the report of evaluation on a small scale with hepatoma cell line and normal people's fetal liver cell system or hepatoma cell line and other tumor cell line antagonism liver cancer scFv.
Utilize display technique of bacteriophage to make up anti-liver cancer phage scFv storehouse, the anti-liver cancer scFv gene that screening makes new advances from the storehouse, the report that domestic useful hepatoma cell line and normal people's fetal liver cell system or hepatoma cell line and other tumor cell line antagonism liver cancer scFv identify, but do not see with multiple liver cancer cell and multiple control cells, people's liver cancer tissue and multiple control tissue and identify the specific report of anti-liver cancer scFv.
Summary of the invention
The objective of the invention is to overcome the problem that exists on the existing liver cancer treatment, a kind of specific anti-human liver cancer phage single-chain antibody HscFv 4-16 is provided.
Another object of the present invention provides the gene of the above-mentioned specific anti-human liver cancer phage single-chain antibody HscFv 4-16 of coding.
Further purpose of the present invention provides the application of above-mentioned specific anti-human liver cancer phage single-chain antibody HscFv 4-16 in preparation treatment liver-cancer medicine.
It is 5.3 * 10 that the present invention utilizes display technique of bacteriophage to make up storage capacity
7Anti-liver cancer phage scFv storehouse, 3 kinds of hepatoma cell line (SMMC7721, Bel-7402 and HepG2) mixing is carried out multi-turns screen to above-mentioned library, obtained the anti-liver cancer scFv of 1 strain, called after scFv4-16, it hangs down 256 times with hepatoma cell line SMMC-7721 and Bel-7402 bonded titre than control cells system (normal people's fetal liver cell is that L-02, stomach cancer cell are SCG7901 and colon carcinoma cell line CaCO2).Further adopt people's liver cancer tissue, multiple control tissue (normal adult hepatic tissue, cirrhotic tissue, chronic hepatitis tissue, stomach organization, colon cancer tissue, human esophageal carcinoma, cancerous lung tissue) to identify its specificity, the result shows that scFv 4-16 has stronger specificity to liver cancer, and has new liver cancer antigen binding site.
(1) structure in anti-human liver cancer phage single-chain antibody storehouse
1. with mixing hepatoma cell line (SMMC-7721 and HepG2) suspension through abdominal injection, immune BALB/c mouse.2. win mouse spleen, extract the total RNA of splenocyte, Poly AT tract with Trizol reagent
@The total RNA of mRNA purification system purifying obtains mRNA.3. be template with mRNA, reverse transcription becomes cDNA.4. be template with cDNA, respectively with the light chain mix primer of antibody amplify the light chain gene (VL) of repertoire antibody, the heavy chain mix primer of antibody amplifies the heavy chain gene (VH) of repertoire antibody.5. with junction fragment Linker both are reassembled into single-chain antibody gene (VL-Linker-VH), i.e. scFv gene.Then, the scFv gene is connected on the pCANTAB 5E carrier.6. electroporation will carry the recon importing e. coli tg1 of scFv gene.7. transformed bacteria is inoculated in the SOBAG flat board, collects bacterium liquid and promptly obtain anti-human liver cancer phage scFv storehouse, its storage capacity is 5.3 * 10
7
(2) screening in anti-human liver cancer phage scFv storehouse
This research adopts two kinds of methods (colony mining method and picking method) at random to screen.
1. use the positive storehouse of colony mining method (Colony lift assay) screening bacterium: anti-human liver cancer phage scFv storehouse bacterium is seeded in the SOBAG flat board, make the storehouse bacterium be the single clonal growth of dispersive, an aseptic micropore (aperture is 0.22 μ m) nitrocellulose membrane is layered on the bacterium face, treat carefully to remove after film is stained with the storehouse bacterium, this is main film.Simultaneously, will mix hepatoma cell line (SMMC7721, Bel-7402 and HepG2) and be coated on the new aseptic micropore nitrocellulose membrane of another piece, make the liver cancer cell coated film.With liver cancer cell coated film (cell faces up) be layered in the SOBAI flat board, the main film (bacterium faces up) that is stained with the storehouse bacterium is layered on the liver cancer cell coated film, two films are performed azimuth mark, and this SOBAI flat board are placed 30 ℃ of incubated overnight.Infection is carried after storehouse bacterium (TG1) on the last layer master film of scFv gene phage stimulated by IPTG, produce solubility scFv, it sees through the liver cancer cell antigen that wraps quilt on film and the following skim and combines taking-up liver cancer cell coated film, add specificity two anti-(promptly anti-scFv-tag monoclonal antibody), with H
2O
2For substrate, DAB are that developer carries out immune color reaction.According to the position of colour developing spot on the liver cancer cell coated film, on main film, find corresponding storehouse bacterium clone, i.e. positive colony.
2. picking method (Randon pick) is screened positive storehouse bacterium at random: with above-mentioned 3 kinds of mixing hepatoma cell line the original storehouse of anti-human liver cancer phage scFv bacterium is carried out 4 and take turns enrichment (i.e. " combination-wash-out-amplification "), get the 4th and take turns the storehouse bacterium and be diluted to suitable concn and be seeded in the SOBAG flat board, make bacteria growing become the single clone of dispersive.Choosing bacterium colony at random, is target antigen with 3 kinds of liver cancer cells, and cell-ELISA method detects positive bacteria.
3. prepare anti-human liver cancer phage scFv: get above-mentioned positive bacteria 200 μ l, add 10ml2 * YT-AG (containing 100 μ g/ml penbritins, 2% glucose) substratum.30 ℃ of shaking culture are to OD
570=0.5.Add M 13K07 helper phage (1 * 10
10Pfu/ml), continue shaking culture 1h.The centrifugal 10min of 1000g, careful abandoning supernatant is used 10ml 2 * YT-AK (containing 100 μ g/ml penbritins, 50 μ g/ml kantlex) substratum to suspend again and is precipitated, and shaking culture is spent the night.Culture is drawn supernatant liquor with the centrifugal 20min of 1000g.In supernatant, add 2ml PEG/NaCl, ice bath 60min, 4000g, 4 ℃ of centrifugal 30min abandon supernatant liquor.With 4ml PBS/1%BSA suspended sediment, promptly obtain anti-human liver cancer phage scFv.
4. anti-human liver cancer phage scFv and the minimum mensuration that combines titre of liver cancer cell antigen: be target antigen with above-mentioned 3 kinds of liver cancer cells respectively, with normal people's fetal liver cell is that L-02, stomach cancer cell are that SCG7901 and colon carcinoma cell line CaCO2 are contrast antigen, with anti-human liver cancer phage scFv be diluted to 1: 2,1: 4,1: 8,1: 16 ..., 1: 16384 titre, cell-ELISA method detects every each titre and combines with above-mentioned cell antigen respectively.Antibody points out its bonding force strong more with antigenic minimum to combine titre low more.Will with hepatoma cell line minimum combine titre than control cells system low more than 128 times the person be considered as specific anti-human liver cancer scFv.This research obtains a strain specific anti-human liver cancer scFv, called after HscFv 4-16, and it hangs down 256 times with hepatoma cell line SMMC-7721 and Bel-7402 bonded titre than L-02, SCG7901 and CaCO2.
5. HscFv 4-16 combines with people's liver cancer tissue: with 62 routine people's liver cancer tissues is target antigen, with 30 routine liver cirrhosis, 30 routine chronic hepatitiss, 20 routine cancer of the stomach, 20 routine colorectal carcinomas, the 20 routine esophageal carcinoma, 20 routine lung cancer and 12 routine normal adult liver organizations is contrast antigen, and employing immunohistochemical methods method detects the reaction to specific HscFv 4-16 of hepatoma cell line and above-mentioned tissue respectively.
6. the amplification of HscFv 4-16 gene: carry the storehouse bacteria plasmid DNA of HscFv 4-16 gene, pcr amplification goal gene with plasmid extraction reagent Rapid Plasmid DNADailyMin-prep Kit extracting.Used upstream primer (P1) is 5 '-CAA CGT GAA AAA ATTATT ATT CGC-3 ', and downstream primer (P2) is 5 '-GTA AAT GAA TTT TCT GTATGA GG-3 '.Reaction conditions is: first 94 ℃ of pre-sex change 3min, 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min then, 30 circulations, last 72 ℃ of 5min.The about 729bp of size of the HscFv4-16 gene that this research amplifies.
7. HscFv 4-16 gene sequencing: this project adopts two-way order-checking with the PCR product order-checking of HscFv 4-16 gene, and the upstream and downstream sequencing primer is identical with the primer of pcr amplification.Measured HscFv 4-16 mrna length is 729bp.
(3) expression of HscFv 4-16: HscFv 4-16 can be expressed in cytoplasm and the culture supernatant of host bacterium, and molecular weight is 31KD.
(4) antihepatocarcinoma effect of HscFv 4-16: this antibody can make the liver cancer cell growth of cultivation slow down, and division reduces mutually, cell rounding, and how spherical in shape or irregular shape, roughen, adherent minimizing, cell colony forms few, and is adherent poor.With the increasing and increase (promptly being dose-dependently) of concentration, inhibiting rate is up to 31.0% to the restraining effect of liver cancer cell growth for it.
Compared with prior art, the present invention has following beneficial effect: the present invention screens 1 specific anti-human liver cancer phage scFv gene from the anti-human liver cancer phage single-chain antibody storehouse that makes up, be HscFv 4-16, the anti-human liver cancer scFv of its expression is to hepatoma cell line (SMMC7721, Bel7402) and people's liver cancer tissue have high specific reaction, minimumly than normal people fetal liver cell be in conjunction with titre, low 256 times of stomach cancer cell system and colon carcinoma cell line, with the positive reaction rate of people's liver cancer tissue be 35.5%, with the positive reaction rate of liver cirrhosis and chronic hepatitis tissue be 6.7%, with the positive reaction rate of cancer of the stomach and colon cancer tissue be 5.0%, with the normal adult hepatic tissue, association reaction does not take place in human esophageal carcinoma and cancerous lung tissue, and the positive rate of liver cancer tissue is apparently higher than other tissue (P＜0.05).Antibody HscFv 4-16 has following industrialization prospect to the high specificity of liver cancer: 1. be developed to immunology research reagent; 2. be developed to the level diagnosis medicine of liver cancer; 3. be developed to the target therapeutic agent of liver cancer; 4. be developed to liver cancer serum and learn diagnostic kit.
Description of drawings
Fig. 1 is the gene order figure of HscFv 4-16;
Fig. 2 is the immunohistochemical methods figure as a result of HscFv 4-16 and mixed type people liver cancer tissue association reaction;
Fig. 3 is the immunohistochemical methods figure as a result of HscFv 4-16 and human hepatocellular carcinoma tissue bond reaction;
Fig. 4 is the immunohistochemical methods figure as a result of HscFv 4-16 and people's cirrhotic tissue association reaction;
Fig. 5 is the immunohistochemical methods figure as a result of HscFv 4-16 and people's chronic hepatitis tissue bond reaction;
Fig. 6 is the immunohistochemical methods result of HscFv 4-16 and normal adult hepatic tissue association reaction;
Fig. 7 is the immunohistochemical methods result of HscFv 4-16 and the reaction of people's adenocarcinoma of stomach tissue bond;
Fig. 8 is the immunohistochemical methods result of HscFv 4-16 and people's esophageal squamous cell carcinoma tissue association reaction;
Fig. 9 is the immunohistochemical methods result of HscFv 4-16 and the reaction of human colon adenocarcinoma's tissue bond;
Figure 10 is the immunohistochemical methods result of HscFv 4-16 and the reaction of human lung adenocarcinoma tissue bond;
Figure 11 is the SDS-polyacrylamide gel electrophoresis result of HscFv 4-16;
Figure 12 is the Westen blotting detected result of HscFv 4-16;
The hepatoma cell line SMMC-7721 growing state figure that Figure 13 handles for HscFv 4-16;
Figure 14 is the hepatoma cell line SMMC-7721 growing state figure that does not handle with HscFv 4-16;
Wherein, among Fig. 1, sequence of blocks: restriction enzyme site; Italic sequence: heavy chain variable region gene (363 base pairs, 121 amino acid of encoding); Underscore sequence: connexon (45 base pairs, 15 amino acid of encoding); Runic sequence: chain variable region gene (321 base pairs, 107 amino acid of encoding); Base pair 729 altogether, 243 amino acid of encoding.Among Fig. 2, the positive result of left figure (is anti-with HscFv 4-16), the visible brown granular in cancer cells surface; The negative contrast of right figure (is anti-with PBS), brown granular is not seen on the cancer cells surface.Among Fig. 3, the positive result of left figure (is anti-with HscFv 4-16), the visible brown granular in cancer cells surface; The negative contrast of right figure (is anti-with PBS), brown granular is not seen on the cancer cells surface.Among Fig. 4, be one anti-with HscFv 4-16, the result that is negative, cell surface is not seen brown granular.Among Fig. 5, be one anti-with HscFv 4-16, the result that is negative, cell surface is not seen brown granular.Among Fig. 6, be one anti-with HscFv 4-16, the result that is negative, cell surface is not seen brown granular.
Among Fig. 7, be one anti-with HscFv 4-16, the result that is negative, brown granular is not seen on the cancer cells surface.Among Fig. 8, be one anti-with HscFv 4-16, the result that is negative, brown granular is not seen on the cancer cells surface.Among Fig. 9, be one anti-with HscFv 4-16, the result that is negative, brown granular is not seen on the cancer cells surface.Among Figure 10, be one anti-with HscFv 4-16, the result that is negative, brown granular is not seen on the cancer cells surface.Among Figure 11,1,2,3 swimming lanes are respectively in the culture supernatant, the HscFv 4-16 in intact cell and the cytoplasm, M: protein standard molecular weight.Among Figure 12,1,2,3 swimming lanes are respectively in the cytoplasm, the HscFv 4-16 in intact cell and the culture supernatant.Among Figure 13, visible cell poor growth under the light microscopic, division reduces mutually, cell rounding, how spherical in shape or irregular shape, roughen, adherent minimizing, cell colony forms few, and is adherent poor.Among Figure 14, under the light microscopic visible cell growth vigorous, be foliated lamellar, the form rule, cell colony is very obvious, and is adherent good.
Embodiment
Embodiment 1
One, the acquisition of HscFv 4-16 gene
1. the structure in anti-human liver cancer phage single-chain antibody storehouse:
1. with mixing hepatoma cell line (SMMC-7721 and HepG2) suspension through abdominal injection, immune BALB/c mouse.2. win mouse spleen, extract the total RNA of splenocyte, Poly AT tract with Trizol reagent
@The total RNA of mRNA purification system purifying obtains mRNA.3. be template with mRNA, reverse transcription becomes cDNA.4. be template with cDNA, respectively with the light chain mix primer of antibody amplify the light chain gene (VL) of repertoire antibody, the heavy chain mix primer of antibody amplifies the heavy chain gene (VH) of repertoire antibody.5. with junction fragment Linker both are reassembled into single-chain antibody gene (VL-Linker-VH), i.e. scFv gene.Then, the scFv gene is connected on the pCANTAB 5E carrier.6. electroporation will carry the recon importing e. coli tg1 of scFv gene.7. transformed bacteria is inoculated in the SOBAG flat board, collects bacterium liquid and promptly obtain anti-human liver cancer phage scFv storehouse, its storage capacity is 5.3 * 10
7
2. the screening in anti-human liver cancer phage scFv storehouse:
1. use the positive storehouse of colony mining method (Colony lift assay) screening bacterium: anti-human liver cancer phage scFv storehouse bacterium is seeded in the SOBAG flat board, make the storehouse bacterium be the single clonal growth of dispersive, an aseptic micropore (aperture is 0.22 μ m) nitrocellulose membrane is layered on the bacterium face, treat carefully to remove after film is stained with the storehouse bacterium, this is main film.Simultaneously, will mix hepatoma cell line (SMMC7721, Bel-7402 and HepG2) and be coated on the new aseptic micropore nitrocellulose membrane of another piece, make the liver cancer cell coated film.With liver cancer cell coated film (cell faces up) be layered in the SOBAI flat board, the main film (bacterium faces up) that is stained with the storehouse bacterium is layered on the liver cancer cell coated film, two films are performed azimuth mark, and this SOBAI flat board are placed 30 ℃ of incubated overnight.Infection is carried after storehouse bacterium (TG1) on the last layer master film of scFv gene phage stimulated by IPTG, produce solubility scFv, it sees through the liver cancer cell antigen that wraps quilt on film and the following skim and combines taking-up liver cancer cell coated film, add specificity two anti-(promptly anti-scFv-tag monoclonal antibody), with H
2O
2For substrate, DAB are that developer carries out immune color reaction.According to the position of colour developing spot on the liver cancer cell coated film, on main film, find corresponding storehouse bacterium clone, i.e. positive colony.
3. the preparation of anti-human liver cancer phage scFv reaches and the minimum mensuration that combines titre of liver cancer cell antigen:
Get above-mentioned positive bacteria 200 μ l, add 10ml 2 * YT-AG substratum.30 ℃ of shaking culture are to OD
570=0.5.Add M13K07 helper phage (1 * 10
10Pfu/ml), continue shaking culture 1h.The centrifugal 10min of 1000g, careful abandoning supernatant is used 10ml 2 * YT-AK substratum to suspend again and is precipitated, and shaking culture is spent the night.Culture is drawn supernatant liquor with the centrifugal 20min of 1000g.In supernatant, add 2ml PEG/NaCl, ice bath 60min, 4000g, 4 ℃ of centrifugal 30min abandon supernatant liquor.With 4ml PBS/1%BSA suspended sediment, promptly obtain anti-human liver cancer phage scFv.(SMMC-7721, HepG-2 and Bel-7402) is target antigen with 3 kinds of hepatoma cell line, with normal people's fetal liver cell is that L-02, stomach cancer cell are that SCG7901 and colon carcinoma cell line CaCO2 are contrast antigen, with anti-human liver cancer phage scFv be diluted to 1: 2,1: 4,1: 8,1: 16 ..., 1: 16384 titre, cell-ELISA method detects every each titre and combines with above-mentioned cell antigen respectively.Antibody points out its bonding force strong more with antigenic minimum to combine titre low more.Will with hepatoma cell line minimum combine titre than control cells system low more than 128 times the person be considered as specific anti-human liver cancer scFv.This research obtains a strain specific anti-human liver cancer scFv, called after HscFv 4-16.The length of the goal gene HscFv 4-16 that obtains is 729bp (Fig. 1).
4.HscFv 4-16 further identifies (adopting the immunohistochemical methods method) to hepatocellular carcinoma tissue-specific:
Paraffin section with liver cancer, chronic hepatitis, liver cirrhosis, cancer of the stomach, colorectal carcinoma, the esophageal carcinoma, cancerous lung tissue places hot air drying oven respectively, 56 ℃ of 2h.Dimethylbenzene dewaxing 2 times, each 10min washes 2 times, 80% ethanol with 95% ethanol successively again and washes 1 time, aquae destillata flushing 1min, and fresh 3% hydrogen peroxide room temperature is washed 5min, to remove non-specific catalase.Wash 2 times, 80% ethanol with 95% ethanol again and wash 1 time, aquae destillata flushing 1min, phosphoric acid buffer (PBS) is washed 2 times, each 1min.Drip the normal mouse serum of dilution in 1: 10, put 10min in the wet box, painted to reduce non-specific background.The unnecessary serum that inclines adds the HscFv 4-16 of 1: 10 titre, and control group adds PBS, puts in the wet box 4 ℃ and spends the night.PBS washes 3 times, and each 1min adds two anti-(1: 1000 dilution HRP/Anti-E Tag Conjugate), puts wet box and spends the night for interior 4 ℃.PBS washes 3 times, and each 1min is with the DAB colour developing 30min that contains 0.05% hydrogen peroxide, flushing with clean water 10min, haematoxylin redyeing 1min, flushing with clean water 10min, with 95% ethanol dehydration, dry up neutral gum mounting, microscopically observation.Immunohistochemical methods is judging criterion as a result: the ratio shared according to positive cell in the tissue slice, the immunohistochemical methods result is divided into 4 grades, promptly divides negative-(positive cell is less than 1%), positive+(positive cell accounts for 1-29%), positive ++ (positive cell accounts for 30-49%) and the positive +++(positive cell accounts for more than 50%).Immunohistochemical methods detects and shows: the positive rate of HscFv 4-16 and people's liver cancer tissue is 35.5%, be 6.7% with the positive reaction rate of liver cirrhosis and chronic hepatitis tissue, be 5.0% with the positive reaction rate of cancer of the stomach and colon cancer tissue, do not react with the esophageal carcinoma, lung cancer, positive hepatic tissue, the positive rate of liver cancer tissue is apparently higher than other tissue (P＜0.05) (Fig. 2～10).
5. contain the extraction of HscFv 4-16 cloned plasmids DNA:
Collect 1ml incubated overnight and contain the bacterium liquid of goal gene in 2 * YT substratum with centrifuge tube, the centrifugal 30s of 12000g abandons most supernatant.The BufferS1 that has added Rnase A1 with the 250 μ l bacterial precipitation that fully suspends.Add 250 μ l Buffer S2, gentle in 5min, spin upside down and mix 4-6 time fully.Add 400 μ l Buffer S3, leniently spin upside down 8-10 time, room temperature leaves standstill 2min.DNA is prepared the centrifuge tube that pipe places 2ml, and above-mentioned sample is centrifugal, and supernatant adds DNA and prepares in the pipe the centrifugal 1min of 2500g.Abandon filtrate, DNA is prepared pipe put back in the former 2ml centrifuge tube, add 500 μ l Buffer W1, the centrifugal 1min of 2500g.Abandon filtrate, DNA is prepared pipe put back in the former 2ml centrifuge tube, add the Buffer W2 that 700 μ l have added dehydrated alcohol, the centrifugal 1min of 2500g washs 1 time with 700 μ l Buffer W2 with same method again.DNA is prepared pipe place the 1.5ml centrifuge tube, the centrifugal 1min of 12000g.DNA is prepared pipe place another clean 1.5ml centrifuge tube, adding 60 μ l temperature in centrifuge tube silica film central authorities is 60 ℃ deionized water, and room temperature leaves standstill 1min, the centrifugal 1min eluted dna of 12000g.
6.HscFv the amplification of 4-16 gene:
Get the plasmid DNA of the above-mentioned HscFv of containing 4-16 gene, pcr amplification goal gene, the primer P1 are 5 '-CAA CGT GAA AAA ATT ATT ATT CGC-3 ', and P2 is 5 '-GTA AAT GAA TTT TCT GTA TGA GG-3 '.Reaction conditions is: first 94 ℃ of pre-sex change 3min, 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min then, 30 circulations, last 72 ℃ of 5min.
7.HscFv the mensuration of 4-16 gene order:
Get the clone bacterium liquid 200 μ l of the above-mentioned HscFv of containing 4-16 gene respectively, add in 10ml2 * YT substratum (containing 100 μ g/ml penbritins), put 30 ℃ of vibrations (250rpm/min) and be cultured to A
570=0.6.Get 1-4ml bacterium liquid, contain the plasmid DNA of HscFv 4-16 gene by above method extracting.Pcr amplification goes out goal gene product to be measured, and amplimer P1 (S1) is 5 '-CAA CGT GAA AAA ATT ATT ATT CGC-3 ', and P2 (S6) is 5 '-GTA AATGAA TTT TCT GTA TGA GG-3 '.The PCR product is served marine life engineering corporation, Shanghai Bo Ya biotech company and the order-checking of Beijing AudioCodes biotech company respectively, and sequencing primer is identical with the PCR primer.
Two, the expression of HscFv 4-16
1. the preparation of cytoplasm and intact cell HscFv 4-16 soluble antibody:
1. preparation contains the antibody library of HscFv 4-16 phage: get the bacterium flat board that coating contains HscFv 4-16 phage, add 5ml 2 * YT substratum, scrape bacterium colony with glass, 37 ℃ of 210rpm jolting 1h, the centrifugal 10min of 2000rpm.With 5ml 2 * YT suspension precipitation, transfer in the sterilization polypropylene test tube of a 50ml volume, being diluted to the OD value is 0.3, calculates final volume, adding penbritin and glucose are 100ug/ml and 2% to final concentration respectively, helper phage 2 * 10
10Pfu, 37 ℃ of jolting 1h.Get 1ml and be added to 10ml 2 * YTAK substratum (containing the penbritin final concentration is 100ug/ml, kantlex 50ug/ml), 37 ℃ of joltings are spent the night.The centrifugal 10min of 2000rpm gets supernatant.
2. prepare intestinal bacteria HB2151: intestinal bacteria HB2151 dry powder is added in 1ml2 * YT substratum 37 ℃ of 210rpm shaken overnight.Get 1 ring overnight culture line with the transfering loop of sterilization and be coated on the microtest plate 37 ℃ of incubated overnight.Picking mono-clonal HB2151 bacterium adds in 5ml 2 * YT substratum on flat board, and 37 ℃ of 210rpm joltings are spent the night.Get 50 μ L bacterium liquid and add in 50ml 2 * YT substratum, 37 ℃ of 210rpm joltings are to OD
600Value is 0.3-0.5.Phage-infect HB2151: in test tube, add the above-mentioned HB2151 bacterium of 400 μ L liquid, add again and contain HscFv 4-16 phage solution 5 μ L, 37 ℃ of 150rpm jolting at intermittence 30min.Coated plate: get 100 μ L bacterium liquid,, get each 100 μ L of stoste and diluent and be coated on the SOBAG-N flat board 30 ℃ of overnight incubation with 2 * YT dilution (1: 10,1: 100,1: 1000).
3. prepare HscFv 4-16: from 5 mono-clonals of the dull and stereotyped picking of SOBAG-N, add respectively in the test tube of 52 * YTAG substratum that contain the 5ml prepared fresh, 30 ℃ of 210rpm joltings are spent the night, and bacterium liquid are added in 2 * YTAG substratum of 50ml prepared fresh 30 ℃ of 210rpm jolting 1h.1500g * 20min is centrifugal, removes supernatant.Precipitation is suspended in 2 * YTAI substratum (not containing glucose) of 50ml prepared fresh, 30 ℃ of 210rpm joltings are spent the night.Bacterium liquid is divided into two pipes, and 1500g * 20min is centrifugal, carefully draws supernatant liquor, filters-20 ℃ of preservations with 0.45 micron filter.One pipe precipitation is used for extracting the HscFv 4-16 soluble antibody of cytoplasm, and another pipe precipitation is used to extract the HscFv4-16 antibody of intact cell.Extract the HscFv 4-16 soluble antibody in the cytoplasm: 1 * TES damping fluid of 500 μ L precoolings (0.2M Tris-HCl pH8.0,0.5mM EDTA and 0.5M sucrose) suspends and precipitates, 0.2 * the TES that adds 750 μ L precoolings shakes up, ice bath 30min, be transferred to another centrifuge tube, the centrifugal 10min of 15000rpm, careful absorption supernatant liquor ,-20 ℃ of preservations.Extract the HscFv 4-16 antibody of intact cell: 500 μ L, 1 * PBS precipitation that suspends, boil 5min, ice bath 30min, the centrifugal 10min of 15000rpm carefully draws supernatant liquor ,-20 ℃ of preservations.
2. the concentrating of HscFv 4-16 in the microbial culture supernatant: the precipitation that adopts the saturated ammonium sulphate method to carry out antibody concentrates.
The beaker that 1. will fill intestinal bacteria HB2151 culture supernatant (containing HscFv 4-16 protein soln) places in another large beaker that ice cube is housed, and stirs on magnetic stirring apparatus.2. in culture supernatant, add ammonium sulfate while stirring slowly to saturated (dissolve 56.8g ammonium sulfate approximately by every 100ml culture supernatant and calculate the ammonium sulfate consumption), in 5～10min, finish this step.3. continue to stir 10～30min.4. the centrifugal 30min of 3000 * g.5. abandoning supernatant is suspended in precipitation in the PBS damping fluid of 1～2 times of throw out volume.6. the centrifugal residual insoluble substance (may be metaprotein etc.) of removing.7. the dialysis tubing of the gained protein soln being packed into, ammonium sulfate is removed in dialysis.Whether 8. detect dialysis with 10% bariumchloride complete.
3.HscFv the SDS-polyacrylamide gel electrophoresis of 4-16 detects:
Adopt the miniature vertical slab electrophoresis groove of Bio-Rad company to prepare the thick polyacrylamide gel of 0.75mm.At first prepare 10ml 12% separation gel (water 3.3ml, 30% acrylamide soln 4ml, 1.5M Tris 2.5ml, 10%SDS 0.1ml, 10% ammonium persulphate 0.1ml, TEMED4 μ l).Encapsulating immediately behind the mixing carefully adds distilled water and covers, polymerized at room temperature 30min, and after the glue polymerization, distilled water inclines.Prepare 5ml 5% spacer gel (water 3.4ml, 30% acrylamide soln 0.83ml, 1.5M Tris 0.63ml, 10%SDS 0.05ml, 10% ammonium persulphate 0.05ml, TEMED 5 μ l) then.Insert sample comb, directly pouring into spacer gel on the polymeric separation gel, avoid producing bubble, polymerized at room temperature 30min.The careful sample comb of taking out is carefully washed the application of sample hole count all over also blotting residuary water with the fine filtering paper slip with distilled water, and gel slab is installed on the electrophoresis chamber, adds 1 * electrophoretic buffer.HscFv 4-16 protein sample and albumen Marker in an amount of cytoplasm, intact cell and the culture supernatant of above-mentioned preparation are gone up sample simultaneously, and institute's making alive is 8V/cm.After the dyestuff forward position enters separation gel, voltage is increased to 15V/cm, continue electrophoresis when tetrabromophenol sulfonphthalein arrives the separation gel bottom, powered-down carefully takes out gel.Gel is carried out coomassie brilliant blue staining according to a conventional method, the gel imaging system shooting.The SDS-polyacrylamide gel electrophoresis shows, all visible special HscFv 4-16 protein band in microbial culture supernatant, cytoplasm and intact cell extract, and molecular weight is about 31KD, sees Figure 11.
4.HscFv the Western blotting of 4-16 detects:
1. SDS-polyacrylamide gel electrophoresis: HscFv 4-16 protein sample in an amount of cytoplasm, intact cell and the culture supernatant is carried out polyacrylamide gel electrophoresis as stated above, finish, carefully take out gel.2. protein sample is transferred on the nitrocellulose filter: in transfer box, place polyacrylamide gel-nitrocellulose filter " sandwich " structure in proper order by fiber mat, filter paper, gel, nitrocellulose filter, filter paper, fiber mat successively, shut transfer box and insert in the electrophoresis chamber 200mA Constant Electric Current transferase 12 h.3. seal nitrocellulose filter: with 10% skimmed milk dry powder PBS room temperature sealing 60min.With PBST (potassium primary phosphate 0.2 gram, Repone K 0.2 gram, Tween200.5ml, sodiumazide 0.2 gram, water 1000ml, pH 7.4 for sodium-chlor 8 gram, Sodium phosphate dibasic 2.9 grams) vibration washing nitrocellulose filter once, PBS washes 3 times, each 5min.4. add anti-Tag-E antibody: drip anti-Tag-E antibody-solutions 10ml (concentration 8ug/ml), hatched under the room temperature 1 hour, PBST vibration washing nitrocellulose filter 1 time, PBS washes 3 times, each 5min, airing.5. add ELIAS secondary antibody: add the enzyme mark sheep anti-mouse antibody 10ml of dilution, 37 degree are hatched 60min.With PBST vibration washing nitrocellulose filter once, PBS washes 3 times, each 5min.6. colour developing: add substrate solution TMB-hydrogen peroxide urea and use liquid A, each 2.5ml of B, behind the mixing nitrocellulose filter is immersed, the colour developing of room temperature lucifuge, colour developing back distilled water flushing stopped reaction, gel imaging system shooting.Westen Blot analyzes demonstration, nitrocellulose filter in transfer printing is equivalent to the 31KD place, the positive staining band of visible three pale bluish greens is respectively the HscFv 4-16 albumen in cytoplasm extract, intact cell extract and the microbial culture supernatant respectively, sees Figure 12.
The anti-liver cancer experiment of embodiment 2HscFv 4-16
(1) experimental technique and process
1. prepare purpose phage antibody HscFv 4-16 in a large number:
1. get and infect the HB2151 bacterium 100 μ L that contain HscFv 4-16 gene phage, add in 2 * YTAG substratum of 100ml prepared fresh 30 ℃ of 210rpm jolting 1h.2. 1500g * 20min is centrifugal, removes supernatant.Precipitation is suspended in 2 * YTAI substratum (not containing glucose) of 100ml prepared fresh, 30 ℃ of 210rpm joltings are spent the night.3. 1500g * 20min is centrifugal, carefully draws supernatant liquor, filters with 0.45 micron filter.4. use ammonium sulfate precipitation filtrate.5. the centrifugal 30min of 3000 * g abandons supernatant liquor, precipitation is suspended in the PBS damping fluid of 1～2 times of throw out volume.6. centrifugally remove residual insoluble substance.7. the dialysis tubing of the HscFv 4-16 protein solution of gained being packed into, ammonium sulfate ,-20 ℃ of preservations are removed in dialysis.
2.HscFv the antihepatocarcinoma effect of 4-16:
(1) light microscopic is observed the influence of HscFv 4-16 to liver cancer cell growth down:
Each hole inoculates 10 respectively in 6 orifice plates
5Individual liver cancer cell SMMC-7721 sets up experimental group and control group separately.Add the concentrated and purified HscFv 4-16 protein product of 1ml in the every 4ml cell culture fluid of experimental group, control group is not done any processing.Cell growth before and after light microscopic is observed down and handled, form, situation such as adherent.
(2) the MTT colorimetry detects the influence of antibody to liver cancer cell growth:
The SMMC-7721 liver cancer cell of 1. taking the logarithm vegetative period, adjusting cell concn with the PRMI1640 nutrient solution is 1 * 10
5/ ml is inoculated in the 96 hole tissue culturing plates, and experimental group and control group are established in 100 μ L/ holes.2. experimental group adds concentrated and purified HscFv 4-16 protein product, it was diluted by 1: 2,1: 4,1: 8,1: 16,1: 32 respectively, and 100 μ L/ holes, control group adds the PRMI1640 nutrient solution of respective volume.Establish 3 multiple holes, 37 ℃, 5%CO for every group
2, saturated humidity is cultivated 48h.3. every hole adds 50 μ L MTT (5mg/ml), continues to cultivate 4h, abandons supernatant liquor, adds DMSO 200 μ L/ holes, 37 ℃ of vibration 10min.4. microplate reader photometry density OD value is a predominant wavelength with 570nm, and 690nm is a reference wavelength.5. inhibiting rate=[1-(experimental group light absorption value/control group light absorption value)] * 100%.6. The data mean ± standard deviation is represented, uses the SPSS11.5 statistical software to handle, and there is statistical significance P＜0.05 for difference.
(2) result
1. among Figure 14, under the light microscopic visible cellular control unit growth vigorous, be foliated lamellar, the form rule, cell colony is very obvious, and is adherent good; Among Figure 13, experimental group cell poor growth, division reduces mutually, cell rounding, how spherical in shape or irregular shape, roughen, adherent minimizing, cell colony forms few, and is adherent poor.
2.HscFv 4-16 is to the influence of liver cancer cell growth: HscFv 4-16 has the effect of obvious inhibition liver cancer cell growth, and with the increase of concentration, inhibiting rate raises gradually, is dose-dependently, inhibiting rate is up to 31.0%, sees Table 1.
The HscFv 4-16 of table 1. different concns is to the influence of SMMC-7721 hepatoma cell proliferation
Annotate: compare * p＜0.05 with control group; * p＜0.01.
Untitled.ST25
SEQUENCE?LISTING
＜110 Ji'nan University
＜120〉specific anti-human liver cancer phage single-chain antibody HscFv 4-16 gene and application thereof
<130>
<160>2
<170>Patent?In?version?3.2
<210>1
<211>742
<212>DNA
＜213〉BALB/c mouse (mus musculus strain balb/c)
<220>
<221>CDS
<222>(14)..(742)
<400>1
ggcccagccg?gcc?atg?gcc?cag?gtg?aag?ctg?cag?cag?gaa?gga?act?gaa 49
Met?Ala?Gln?Val?Lys?Leu?Gln?Gln?Glu?Gly?Thr?Glu
1 5 10
gtg?gta?aag?cct?ggg?gct?tca?gtg?aag?ttg?tcc?tgc?aag?gct?tct?ggc 97
Val?Val?Lys?Pro?Gly?Ala?Ser?Val?Lys?Leu?Ser?Cys?Lys?Ala?Ser?Gly
15 20 25
tac?atc?ttc?aca?agt?tat?gat?ata?gac?tgg?gtg?agg?cag?acg?cct?gaa 145
Tyr?Ile?Phe?Thr?Ser?Tyr?Asp?Ile?Asp?Trp?Val?Arg?Gln?Thr?Pro?Glu
30 35 40
cag?gga?ctt?gag?tgg?att?gga?tgg?att?ttt?cct?gga?gag?ggg?agt?act 193
Gln?Gly?Leu?Glu?Trp?Ile?Gly?Trp?Ile?Phe?Pro?Gly?Glu?Gly?Ser?Thr
45 50 55 60
gaa?tac?aat?gag?aag?ttc?aag?ggc?agg?gcc?aca?ctg?agt?gta?gac?aag 241
Glu?Tyr?Asn?Glu?Lys?Phe?Lys?Gly?Arg?Ala?Thr?Leu?Ser?Val?Asp?Lys
65 70 75
tcc?tcc?agc?aca?gcc?tat?atg?gag?ctc?act?agg?ctg?aca?tct?gag?gac 289
Ser?Ser?Ser?Thr?Ala?Tyr?Met?Glu?Leu?Thr?Arg?Leu?Thr?Ser?Glu?Asp
80 85 90
tct?gct?gtc?tat?ttc?tgt?gct?aga?ggg?gac?tac?tat?agg?cgc?tac?ttt 337
Ser?Ala?Val?Tyr?Phe?Cys?Ala?Arg?Gly?Asp?Tyr?Tyr?Arg?Arg?Tyr?Phe
95 100 105
gac?ttg?tgg?ggc?caa?ggg?acc?acg?gtc?acc?gtc?tcc?tca?ggt?gga?ggc 385
Asp?Leu?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Gly?Gly?Gly
110 115 120
ggt?tca?ggc?gga?ggt?ggc?tct?ggc?ggt?ggc?gga?tct?gac?att?gag?ctc 433
Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Asp?Ile?Glu?Leu
125 130 135 140
acc?cag?tct?cca?gca?atc?atg?tct?gca?tct?cca?ggg?gag?agg?gtc?acc 481
Thr?Gln?Ser?Pro?Ala?Ile?Met?Ser?Ala?Ser?Pro?Gly?Glu?Arg?Val?Thr
145 150 155
atg?acc?tgc?agt?gcc?agc?tca?agt?ata?cgt?tac?ata?tat?tgg?tac?caa 529
Met?Thr?Cys?Ser?Ala?Ser?Ser?Ser?Ile?Arg?Tyr?Ile?Tyr?Trp?Tyr?Gln
160 165 170
cag?aag?cct?gga?tcc?tcc?ccc?aga?ctc?ctg?att?tat?gac?aca?tcc?aac 577
Gln?Lys?Pro?Gly?Ser?Ser?Pro?Arg?Leu?Leu?Ile?Tyr?Asp?Thr?Ser?Asn
175 180 185
gtg?gct?cct?gga?gtc?cct?ttt?cgc?ttc?agt?ggc?agt?ggg?tct?ggg?acc 625
Val?Ala?Pro?Gly?Val?Pro?Phe?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr
190 195 200
Untitled.ST25
tct?tat?tct?ctc?aca?atc?aac?cga?atg?gag?gct?gag?gat?gct?gcc?act 673
Ser?Tyr?Ser?Leu?Thr?Ile?Asn?Arg?Met?Glu?Ala?Glu?Asp?Ala?Ala?Thr
205 210 215 220
tat?tac?tgc?cag?gag?tgg?agt?ggt?tat?ccg?tac?acg?ttc?gga?ggg?ggg 721
Tyr?Tyr?Cys?Gln?Glu?Trp?Ser?Gly?Tyr?Pro?Tyr?Thr?Phe?Gly?Gly?Gly
225 230 235
acc?aag?ctg?gag?ctg?aaa?cgg 742
Thr?Lys?Leu?Glu?Leu?Lys?Arg
240
<210>2
<211>243
<212>PRT
＜213〉artificial sequence
<400>2
Met?Ala?Gln?Val?Lys?Leu?Gln?Gln?Glu?Gly?Thr?Glu?Val?Val?Lys?Pro
1 5 10 15
Gly?Ala?Ser?Val?Lys?Leu?Ser?Cys?Lys?Ala?Ser?Gly?Tyr?Ile?Phe?Thr
20 25 30
Ser?Tyr?Asp?Ile?Asp?Trp?Val?Arg?Gln?Thr?Pro?Glu?Gln?Gly?Leu?Glu
35 40 45
Trp?Ile?Gly?Trp?Ile?Phe?Pro?Gly?Glu?Gly?Ser?Thr?Glu?Tyr?Asn?Glu
50 55 60
Lys?Phe?Lys?Gly?Arg?Ala?Thr?Leu?Ser?Val?Asp?Lys?Ser?Ser?Ser?Thr
65 70 75 80
Ala?Tyr?Met?Glu?Leu?Thr?Arg?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr
85 90 95
Phe?Cys?Ala?Arg?Gly?Asp?Tyr?Tyr?Arg?Arg?Tyr?Phe?Asp?Leu?Trp?Gly
100 105 110
Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly
115 120 125
Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Asp?Ile?Glu?Leu?Thr?Gln?Ser?Pro
130 135 140
Ala?Ile?Met?Ser?Ala?Ser?Pro?Gly?Glu?Arg?Val?Thr?Met?Thr?Cys?Ser
145 150 155 160
Ala?Ser?Ser?Ser?Ile?Arg?Tyr?Ile?Tyr?Trp?Tyr?Gln?Gln?Lys?Pro?Gly
165 170 175
Ser?Ser?Pro?Arg?Leu?Leu?Ile?Tyr?Asp?Thr?Ser?Asn?Val?Ala?Pro?Gly
180 185 190
Val?Pro?Phe?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Ser?Tyr?Ser?Leu
195 200 205
Thr?Ile?Asn?Arg?Met?Glu?Ala?Glu?Asp?Ala?Ala?Thr?Tyr?Tyr?Cys?Gln
210 215 220
Untitled.ST25
Glu?Trp?Ser?Gly?Tyr?Pro?Tyr?Thr?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Glu
225 230 235 240
Leu?Lys?Arg
Claims (5)
1. specific anti-human liver cancer phage single-chain antibody HscFv 4-16, its aminoacid sequence is shown in SEQ IDNO:2.
2. the gene of the described specific anti-human liver cancer phage single-chain antibody HscFv of the claim 1 of an encoding 4-16, its nucleotide sequence is shown in SEQ ID NO:1.
3. the recombinant vectors that contains the described gene of claim 2.
4. the recombinant microorganism that transforms of the described recombinant vectors of claim 3.
5. the application of the described specific anti-human liver cancer phage single-chain antibody HscFv of claim 1 4-16 in preparation treatment liver-cancer medicine.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1241574A (en) * | 1998-07-02 | 2000-01-19 | 中国人民解放军第四军医大学 | Liver cancer-resisting monoclone antibody HAb25 and its single stranded antibody and bifunctional antibody |
| US6083915A (en) * | 1991-05-10 | 2000-07-04 | Biomeasure, Inc. | Method for treating liver cancer |
| CN1381461A (en) * | 2002-03-15 | 2002-11-27 | 陈志南 | Light chain or heavy chain variable region gene of human liver cancer monoclonal antibody HAb18 and its application |
| CN1535985A (en) * | 2003-04-09 | 2004-10-13 | 中国人民解放军第四军医大学 | Genetically-engineered single chain antibody scFv 25 for resisting liver cancen |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6083915A (en) * | 1991-05-10 | 2000-07-04 | Biomeasure, Inc. | Method for treating liver cancer |
| CN1241574A (en) * | 1998-07-02 | 2000-01-19 | 中国人民解放军第四军医大学 | Liver cancer-resisting monoclone antibody HAb25 and its single stranded antibody and bifunctional antibody |
| CN1381461A (en) * | 2002-03-15 | 2002-11-27 | 陈志南 | Light chain or heavy chain variable region gene of human liver cancer monoclonal antibody HAb18 and its application |
| CN1535985A (en) * | 2003-04-09 | 2004-10-13 | 中国人民解放军第四军医大学 | Genetically-engineered single chain antibody scFv 25 for resisting liver cancen |
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