CN102250239A - Protein capable of combining with vp60 protein of rabbit hemorrhagic disease virus and use thereof - Google Patents
Protein capable of combining with vp60 protein of rabbit hemorrhagic disease virus and use thereof Download PDFInfo
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Abstract
The invention discloses a protein capable of combining with vp60 protein of rabbit hemorrhagic disease virus and use thereof. The protein is characterized by at least containing a sequence represented by SEQ ID No.1. In the invention, the protein 215M22 kappa1 which can interact with the vp60 protein of rabbit hemorrhagic disease virus is fished out from a rabbit liver tissue cDNA library by a yeast twohybrid technique. Through coimmunoprecipitation tests, the protein is proved in vitro to be capable of combining with virions, and purified kappa 1 protein can inhibit the agglutination effect of viral antigens on human 'O' type blood cells. After the protein is inoculated into an RHDV antibody negative rabbit together with the rabbit hemorrhagic disease virus for 8 days, the rabbit is still alive and appears healthy, and the results of pathological anatomy and biopsy indicate the organs are normal. The protein kappa1 can be used in the preparation of medicines for treating rabbit hemorrhagic disease.
Description
Technical field
The present invention relates to a kind of protein and application thereof that can mutually combine with rabbit hemorrhagic disease virus coat protein VP60, particularly a kind of kappa1 light chain immunoglobulin (Ig), and the application in preparation treatment rabbit haemorrhagic disease medicine belong to biomedicine field.
Background technology
Rabbit haemorrhagic disease (Rabbit Hemorrhagic Disease, RHD), be commonly called as " rabbit pest " (Rabbit Plague), be by rabbit hemorrhagic disease virus (Rabbit Hemorrhagic Disease Virus, RHDV) acute, strong, the contagious disease that cause, be changed to principal character with hepatic necrosis, substantial viscera oedema, respiratory system and many organs of whole body extravasated blood and hemorrhagic, rabbit keeping is caused destructive strike usually.VP60 is as unique coat protein of virus, goes through the popular of strain more than 20 year, still keeping the homology of height, and it has important effect aspect infection of disease and the host immune.
Virus is a large amount of fast breedings in liver, cause the generation of lethality hepatitis, show and exist multiple in the liver organization and the interactional albumen of rabbit hemorrhagic disease virus, therefore the interaction protein of screening rabbit hemorrhagic disease virus VP 60 albumen from rabbit liver, significant at aspects such as the prevention of this disease, treatments.
Summary of the invention
One of technical problem to be solved by this invention provides a kind of protein or nucleic acid that can be used for preparing the rabbit haemorrhagic disease medicine, this albumen or nucleic acid can combine the purpose that reaches neutralization virus with rabbit hemorrhagic disease virus VP 60 albumen or nucleotide sequence, with a kind of albumen that mutually combines with rabbit hemorrhagic disease virus VP 60 albumen of the present invention, it is characterized in that comprising at least the sequence shown in SEQ ID NO:1;
Find that through order-checking and sequence alignment analysis this albumen and kappa1 light chain immunoglobulin (Ig) have higher homology, and still kappa1 light chain immunoglobulin (Ig) is not used for the treatment of the report of rabbit haemorrhagic disease at present;
Preferably, described albumen is 215M22 kappa1 light chain immunoglobulin (Ig), Genbank accession number AY495826.1;
Preferred, described Argine Monohydrochloride sequence is shown in SEQ ID NO:1.
The present invention also provides coding the above proteic nucleotide sequence;
Preferably, described nucleotide sequence is shown in SEQ ID NO:2;
The invention still further relates to the recombinant vectors that contains described nucleotide sequence, the host bacterium that contains described recombinant vectors, the host bacterium can be intestinal bacteria (colon bacillus, Escherichia coli) BL21 (DE3).
Two of technical problem to be solved by this invention provides a kind of pharmaceutical composition, comprises the described albumen of effective dose and the carrier of pharmaceutically accepting; And
A kind of pharmaceutical composition comprises the described nucleotide sequence of effective dose and the carrier of pharmaceutically accepting;
Three of technical problem to be solved by this invention provides the application of above-described albumen in preparation treatment rabbit haemorrhagic disease medicine; And
Described nucleotides sequence is listed in the application in the preparation treatment rabbit haemorrhagic disease medicine.
Technical problem to be solved by this invention realizes by the following method:
1, from rabbit liver tissue cDNA library, angles and obtain interactional albumen with rabbit hemorrhagic disease virus structural protein VP60
In this research,, from rabbit liver tissue cDNA library, angle and obtain interactional albumen with rabbit hemorrhagic disease virus structural protein VP60 by yeast-two hybrid technique.Find that through order-checking and sequence alignment analysis this albumen and kappa1 light chain immunoglobulin (Ig) have higher homology, are that it is a 215M22 kappa1 light chain immunoglobulin (Ig) specifically, Genbank accession number AY495826.1.
2,215M22 kappa1 segmental clone of proteic main encoding gene and expression
The proteic main encoding gene sheet segment length 480bp of clone 215M22 kappa1, this fragment is connected among the expression vector pET32a, obtain recombinant plasmid pKAPPA-1, transformed into escherichia coli BL21 (DE3), this bacterial classification (BL21 (DE3) pKAPPA-1) is colon bacillus (Escherichia coli), be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, the address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, culture presevation is numbered: CGMCC No.4680, the culture presevation time is: on March 16th, 2011.Obtained reorganization kappa1 albumen through abduction delivering.The long 159aa of this proteic encoding amino acid sequence, the about 17kD of molecular weight.
3,215M22 kappa1 albumen and the external checking of virus particle bonded
Test by co-immunoprecipitation, the external 215M22 kappa1 albumen of having verified has the function that mutually combines with virus particle, the 215M22 kappa1 albumen of purifying can suppress the agglutination of the virus antigen of 8 HAUs to people " O " type erythrocyte under the condition of 0.43 μ g.
4, KAPPA1 prion neutralization test
Count with this protein and 16 sesquialters in the RHDV room temperature of lethal dose and hatch 60min, oral administration, collunarium inoculation RHDV negative antibody rabbit, rabbit still survives after 8 days, appearance health, each internal organs of pathological anatomy and cut sections for microscopic examination are also no abnormal.The virus infection rabbit, dead in 3 days.This protein 21 5M22 kappa1 can be used for the control of the infection of RHDV.
Description of drawings
Fig. 1 is the blood clotting inhibition effect of reorganization 215M22 kappa1 albumen to RHDV;
Fig. 2 is the pathology section examination result;
Fig. 3 is the hemagglutination test result;
Fig. 4 inserts segmental agarose gel electrophoresis figure for rabbit liver yeast cDNA library;
The 1-20:PCR product; M:DL2000 Marker
Fig. 5 is that the PCR of recombinant plasmid pGBKT7-VP60 identifies figure;
1:DL10,000Marker; The PCR of 2:pGBKT7-VP60 identifies; 3: the water contrast
Fig. 6 cuts evaluation figure for the enzyme of recombinant plasmid pGBKT7-VP60;
1: enzyme is cut pGBKT7-VP60 plasmid product; 2:DL10,000Marker
Fig. 7 is fusion product growing state on the QDO flat board of Y187 (pGBKT7-VP60) and AH109 (pGADT7);
A: positive control; B: positive control (QDO-X-α-gal); C: negative control; D: fusion product
Fig. 8 is the screening of 1 yeast two-hybrid positive colony;
Fig. 9 is the main encoding gene electrophorogram of pcr amplification rabbit immunoglobulin 215M22 kappa1 light chain;
Figure 10 cuts qualification result for recombinant plasmid pKAPA-1 through the EcoRI+XhoI enzyme;
Figure 11 is expression in escherichia coli reorganization 215M22 kappa1 protein electrophoresis figure (A: contrast negative bacterium, B: the kappa1 albumen of expression);
Figure 12 is RHDV virus particle purification result (A: electron microscopic observation B:SDS-PAGE gel electrophoresis);
Figure 13 be the immunoprecipitation test-results (1, protein marker; 2, CO-IP result; 3, negative control; 4, positive control).
Embodiment
Below by pharmacological and clinical observation experiment and the present invention will be further described in conjunction with the embodiments, it should be understood that these embodiment only are used for the purpose of illustration, never limit protection scope of the present invention.
The RHDV virus particle is as having the erythrocytic characteristic of aggegation human blood " O " type, virus particle with 0.43 μ g reorganization 215M22 kappa1 albumen and 8 HAUs, incubated at room 45min, the hemagglutination activity that can suppress virus particle, and control group BSA does not have blood clotting inhibition activity, result such as table 1, shown in Figure 1.
Table 1 reorganization 215M22kappa1 albumen suppresses to tire (unit: μ g) to the blood clotting of RHDV
Get 10 RHDV negative antibody rabbits, be divided into two groups immediately, 5 every group.First group of neutralization group: 16 sesquialters are counted the RHDV virus particle of lethal dose (LD50), mix with 40 μ g reorganization 215M22 kappa1 albumen, behind this mixture incubated at room 60min, through the oral cavity, collunarium inoculates.Second group of negative control group: through the oral cavity, collunarium direct inoculation 16 sesquialters count the RHDV virus particle of lethal dose (LD50).In 8 days observation period, 5 rabbits of neutralization group all survive, and appearance health compel to be killed after each internal organs of pathological anatomy and cut sections for microscopic examination also no abnormal (as shown in Figure 2), and it is 2 that the people of liver organization lapping liquid " O " hemaglutination is tired
0, be judged to feminine gender.5 rabbits of negative control group died off in 3 days, and the people of liver organization lapping liquid " O " hemaglutination is tired and is 10log2, the positive, and the result is as shown in table 2, and the hemagglutination test result is as shown in Figure 3.
Table 2 is attacked poison back result
Experimental example 1 angles from rabbit liver tissue cDNA library obtains the interactional albumen with rabbit hemorrhagic disease virus structural protein VP60
The yeast two-hybrid method is angled and is got and the interactional albumen of rabbit hemorrhagic disease virus VP 60 albumen, and used Oligonucleolide primers in this research is as shown in table 3:
| The primer title | Primer sequence |
| The VP60 upstream primer | 5`-CCGGAATTCATGGAGGGCAAAACCCGCACA-3`(EcoR I) |
| The VP60 downstream primer | 5`-ACGCGTCGACTTATCAGACATAAGAAAAGC-3`(Sal I) |
| CDSIII Primer | 5′-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T)30VN-3′)* |
| SMARTIII Oligo | 5′-AAGCAGTGGTATCAACGCAGAGTGGCCATTATGGCCGGG-3′) |
| 5′ |
5′-TTCCACCCAAGCAGTGGTATCAACGCAGAGTGG-3′ |
| 3′ |
5′-GTATCGATGCCCACCCTCTAGAGGCCGAGGCGGCCGACA-3′ |
| AD-5`primer | 5`-CTATTCGATGATGAAGATACCCCACCAAACCC-3` |
| AD-3` |
5`-GTGAACTTGCGGGGTTTTTCAGTATCTACGAT-3` |
1 method
1.1RNA extraction
Adopt Qiagen RNA to extract test kit and extract rabbit hepatic tissue RNA, RNA is detected RNA purity with ultraviolet spectrophotometer after 2 times of the RNase-free water dilutions, read OD260, OD280, OD230, OD260/OD280 ratio, OD260/OD230 ratio and RNA concentration numerical value.Native gel electrophoresis is checked the integrity of RNA.
1.2ds cDNA's is synthetic
According to the cDNA library construction of Clonteh company and synthesizing of the explanation carrying out respectively of screening reagent the box first chain cDNA and double-stranded cDNA, get the agarose gel electrophoresis that 5 μ L PCR products carry out 1.2% concentration.
1.3cDNA purifying and recovery
The cDNA purification column is put upside down resuspended gel matrix for several times, keep purification column upright, break and remove end, put into the 2ml collection tube, remove top cover, the centrifugal 5min of 700g is with level pad balance matrix.Purification column is put into a new 1.5ml centrifuge tube, double-stranded cDNA (ds cDNA) is added the substrate plane center, the centrifugal 5min of 700g, liquid is the ds DNA of purifying in the centrifuge tube of collection.
1.4 the acquisition in yeast library and library quality evalution
1.4.1ds transforming AH109 on a large scale, cDNA prepares the library
1) in the centrifuge tube of an aseptic 15mL, mix following composition (all wanting mixing behind a kind of composition of every adding):
2) add 2.5mL and now join PEG/LiAC solution, place 45min for 30 ℃.Every 15min mixing once;
3) add the aseptic DMSO of 160 μ L, mixing, 42 ℃ of water-bath 20min.Every 10min mixing once;
4) the centrifugal 5min of 700g abandons supernatant, and precipitation is resuspended in the 3mL YPD Plus liquid nutrient medium;
5) 30 ℃, 240r/min shaking culture 90min, the centrifugal 5min of 700g;
6) abandon supernatant, precipitate resuspended resuspended to 15mL 0.9%NaCl.
1.4.2 the cultivation in yeast library, recovery and evaluation
1) gets each 200 μ L of converted product and evenly be applied on the 150mm SD/-Leu plate shared about 100 plates;
2) respectively get 1: 10,1: 100,1: 1000 of 100 μ L and 1: 10000 dilution sterilised yeast suspension evenly is applied on 2 100mm SD/-Leu plates;
3) cultivate 3-6d for 30 ℃, wait for that the clone grows.Count the mono-clonal number on each extent of dilution flat board,, have only when transformation efficiency to be higher than 1 * 10 to calculate transformation efficiency
6Could collect the library during transformant/3 μ g pGADT7-Rec and be used for screening;
Transformation efficiency=(bacterium colony number * total be coated with plate bulk)/(dilution parameters * this plate is coated with plate bulk)
4) the 150mm plate is put 4 ℃ of cooling 3-4hr;
5) add the freezing substratum of 5mL precooling to each plate;
6) with glass stick the bacterium colony on the plate is scraped down, again with pipettor repeatedly pressure-vaccum several times the bacterium colony on the plate is all washed (discarding the zone that mould produces when collecting bacterium colony);
7) washing lotion of 100 plates is collected one, mix;
8) calculate cell density with globulimeter, formula is as follows:
Cell density=microscopically cell count/4 * 10
4* extension rate
If cell density is less than 2 * 10
7Cells/mL, then centrifugal back reaches 2 * 10 with the resuspended cell density that makes of less freezing substratum
7More than the cells/mL.After meeting the requirements, cell density is distributed into the every pipe of 1mL ,-70 ℃ of preservations;
9) will draw 1 μ L library stoste, and add and to contain in the 1.5mL centrifuge tube of 1mL 0.9%NaCl, mixing gently, this is diluent A (Dilu.A, 1: 10
3).By drawing 1 μ L among the Dilu.A, add again among the 1mL 0.9%NaCl, mixing gently, this is diluent B (Dilu.B, 1: 10
6).Be diluted to 200 μ L by absorption 20 μ L among the Dilu.B and be designated as diluent C (Dilu.C, 1: 10
7), diluent A, B and C on average are coated with on 2 SD/-Leu flat boards.30 ℃ of inversions are cultured to bacterium colony and grow, and calculate the dull and stereotyped mono-clonal colony number of going up, and determine cDNA library titre to be amplified:
Library titre (cfu/mL)=(average colony number/bed board volume) * extension rate
10) evaluation of clip size is inserted in the library
20 yeast clones of picking join respectively in the SD/-Leu liquid nutrient medium from the library at random, after about 24hr is cultivated in 30 ℃ of joltings, get yeast liquid as pcr template, and system is as follows:
Reaction parameter: 94 ℃ of 7min; 94 ℃ of 30s, 68 ℃ of 3min, 30 circulations; 68 ℃ of 3min.Get 10 μ L after the PCR EP (end of program) and carry out 1.2% agarose gel electrophoresis.
11) the library est sequence obtaining and analyzing
50 positive colonies of random choose, extract yeast plasmid after pcr amplification insert fragment, will send the order-checking of Invitrogen company behind the purpose fragment purification, sequencing result is removed carrier sequence and inaccurate base after login NCBI website carry out the Blastn comparison.
1.5 the structure of bait plasmid pGBKT7-VP60 and evaluation
Plasmid pMD18-T-VP60 (a) advances pcr amplification as template, reaction parameter in 1: 100 dilution back: 94 ℃ of 5min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 2min, totally 30 circulations; 72 ℃ of 10min, the PCR product is connected to yeast shuttle expression carrier pGBKT7 behind EcoR I+Sal I double digestion, among the transformed into escherichia coli DH5 α.Picking reorganization bacterium colony, through PCR and enzyme cut identify correct after, serve extra large Invitrogen company and carry out sequencing, and with sequencing result with utilizing the NCBI online tool to analyze: http://www.ncbi.nlm.nih.gov/Blast/.
1.6 structure and the evaluation of yeast two-hybrid bait carrier Y187 (pGBKT7-VP60)
1.6.1pGBKT7-VP60 transform Y187
Get a 1.5mL centrifuge tube, add 0.1 μ g pGBKT7-VP60 plasmid DNA and 0.1mg Herring testis carrier DNA, mixing, transformed yeast Y187.Extract test kit with the omega yeast plasmid and extract yeast plasmid, among the transformed into escherichia coli competent cell DH5 α, extract escherichia coli plasmid, enzyme is cut and PCR identifies.
1.6.2 the active detection of bait self activation
To line SD/-Trp/X-α-Gal respectively through the Y187 that contains bait plasmid (pGBKT7-VP60) yeast of identifying, SD/-His/-Trp/X-α-Gal, SD/-Ade/-Trp/X-α-Gal flat board cultivates 3-5d for 30 ℃.Observation contains the Y187 growing state of bait plasmid, judges that can bait start the expression of reporter gene separately.
1.6.3 the toxic detection of bait protein
Picking has changed the yeast Y187 (pGBKT7) of pGBKT7 empty carrier over to respectively, 24hr is cultivated in 240r/min jolting in the SD/-Trp liquid nutrient medium with the single bacterium colony of yeast Y187 (pGBKT7-VP60) that has changed bait plasmid over to, detects the A600 value and carries out the comparison of the speed of growth.
1.7 library screening
1.7.1 yeast mating
Picking is fresh contain recon yeast Y187 (pGBKT7-VP60) bacterium colony (＞2mm), with moving to behind the abundant mixing of 1mL SD/-Trp in the Erlenmeyer flask of the 250mL capacity that contains 50mL SD/-Trp, after 20hr is cultivated in 30 ℃ of 240r/min joltings, survey its A600 value, and yeast cell is counted with the red blood cell count(RBC) plate; With 1mL AH109 yeast library (〉=2 * 10
7Cells) room-temperature water bath is melted; With 5mLY187 bait bacterium (〉=1 * 10
9Cells/mL) join in the aseptic 2L flask mixing with the AH109 yeast library of having melted; Add 45mL2 * YPDA/Kan (50 μ g/mL), mixing; 30 ℃, 50r/min cultivates about 20-24hr; Behind the mating 20hr, microscopically is observed the combination in the mating liquid, and continues to cultivate 4hr; Mating liquid moves in the aseptic centrifuge tube, and 1,000g * 10min abandons supernatant; With the resuspended precipitation of washings behind 2 * YPDA/Kan (50 μ g/mL) flushing Erlenmeyer flask twice (25mL/ time); 1, the centrifugal 10min of 000g abandons supernatant; Precipitation is resuspended with 10mL 0.5 * YPDA in the centrifuge tube.
1.7.2 express the diplontic screening of interaction protein
1) from the mating product, gets 12 μ L by serial dilution in 1: 10,1: 1000,1: 10000, each extent of dilution is got 100 μ L respectively and is seeded to SD/-Trp, SD/-Leu, SD/-Trp/-Leu flat board to calculate mating efficient, to remain the resuspended liquid of about 11mL and be seeded to 150mm QDO flat board, and be inverted in and continue in 30 ℃ of constant incubators to cultivate by each dull and stereotyped 200 μ L; After treating that clone on the flat board grows, the colony number on counting SD/-Trp, SD/-Leu, the SD/-Trp/-Leu flat board is to calculate mating efficient;
Vigor (cfu/mL)=clone's number/(being coated with plate bulk * dilution parameters)
2) vigor on SD/-Leu is an AH109 gamete vigor; Vigor on SD/-Trp is a Y187 gamete vigor; At the SD/-Leu/-Trp vigor is the diploid vigor; Mating efficient=every milliliter of diploid clone number/every milliliter of restriction part clone number * 100%
3) about 1mm that draws at the dull and stereotyped back side of QDO-X-α-Gal
2The cross grid, with Ade
+, His
+Male clone (the albicans Saccharomyces bacterium colony of＞2mm) transfers to respectively and carries out blue hickie screening in the grid on QDO-X-α-Gal flat board;
4) pick out blue yeast colony and be inoculated in once more on QDO-X-α-Gal flat board, and numbering;
5) screen the male colony lift in the QDO liquid nutrient medium with twice, extract yeast plasmid after about 24hr are cultivated in 30 ℃ of joltings;
6) with the yeast plasmid be template, sequence is that primer carries out pcr amplification on the pGADT7-Rec carrier;
1.7.3 the recovery of yeast two-hybrid system checking
The library plasmid that sifts out in the experimentation with the front is transformed into the Y187 yeast, verify its self activation activity and toxic action, simultaneously bait recombinant plasmid pGBKT7-VP60 is transformed in the AH109 yeast, again both is carried out one to one the yeast mating test with checking positive colony and the proteic interaction of VP60.
1.7.4 reply positive colony order-checking and bioinformatic analysis after the checking
The positive plasmid that the recovery of yeast two-hybrid system is verified is served the Hai Shenggong order-checking.Through order-checking, check the order-checking collection of illustrative plates through 23 clones of checking, sequencing sequence is surpassed the incorrect base that 800bp and PloyA sequence occur later remove, obtain effective ESTs sequence thereby remove two ends carrier sequence with DNAStar software in addition.With the Seqman II in the DNAStar software positive colony that filters out is carried out classification analysis, have only a gene normalizing group's numbering to represent that representing with contig of two above sequences arranged with this clone.Utilize Blastx to carry out homology analysis, comparison (http://www.ncbi.nlm.nih.gov/BLAST/) sequence after sorting out, comparison database is mainly all the nonredundancy sequences among Genbank, EMBL, the DDBJ.
2 results
2.1 the extraction of liver organization RNA
The A260/A280 value of total RNA is 2.061, in criterion (A260/A280=1.9-2.1) scope of RNA purity; A260/A230=2.18 in standard range (A260/A230=2.0-2.5), illustrates that thus contained residual salinity is less among the RNA that is extracted.Spectrophotometric RNA concentration is 216 μ g/mL.
Non-sex change agarose gel electrophoresis result shows that 28S rRNA, 18S rRNA band become clear, and the ratio of its brightness is greater than 2.0, and the 5S band almost be cannot see almost not degraded of explanation RNA.The total tissue RNA that shows extraction is more complete, meets requirement of experiment.
2.2ds the synthetic and purifying of cDNA
The total tissue RNA of extracting method with LD-PCR after reverse transcription is synthesized ds cDNA, ds cDNA utilization purification column is removed wherein contained impurity such as enzyme, remove the following fragment of 200bp simultaneously, 1.2% agarose gel electrophoresis shows that the ds cDNA library disperse behind the purifying is distributed between about 0.2-2.5kb, there is intensive band in zone near 1kb, and this section is the high abundance expressing gene.
2.3 library total cellular score and library titre
With glass stick all bacterium colonies are collected together from flat board, about altogether 300mL, with red blood cell count(RBC) plate counting, 10-4 extent of dilution visible cell number is 39 behind the mixing, according to red blood cell count(RBC) plate Counting Formula:
Cell density=microscopically cell count/4 * 10
4* extension rate=39/4 * 10
4* 10
4=9.75 * 10
8Cells/mL
Draw 100 μ L library diluents and be coated on the SD/-Leu flat board, after 3 days visible 10
-7The bacterium colony number is respectively 24 and 30 clones on the flat board, then:
Library titre (cfu/mL)=(colony number/bed board volume) * extension rate=(27cfu/0.1mL) * 10
-7=2.7 * 10
9Cfu/mL
2.4 the clip size analysis is inserted in the library
Positive colony on 20 SD/-Leu flat boards of random choose, with AD-5`primer and AD-3`primer is that primer carries out pcr amplification, 1.2% agarose gel electrophoresis shows: the clone of picking nearly all has the segmental existence of external source, the insertion fragment is different in size, mainly be distributed in 0.5-2.0kb, about average about 1.0kb, recombination fraction was about for 100% (as shown in Figure 4).
2.5 the structure of bait plasmid and evaluation
After the VP60 fragment that the recovery enzyme is cut, be connected on the pGBKT7 carrier that same enzyme cuts, extract the positive pGBKT7-VP60 plasmid of PCR, enzyme is cut and can be produced about 1800bp and 7300bp two bands, and order-checking shows with correct reading frame construction in carrier pGBKT7 (as Fig. 5, shown in Figure 6).
2.6Y187 (pGBKT7-VP60) with the fusion of AH109 (pGADT7)
Y187 (pGBKT7-VP60) and AH109 (pGADT7) are carried out not seeing yeast growth on a small amount of yeast post-coitum QDO flat board, simultaneously, negative control is not seen colony growth, positive control has circular white colony to produce on the QDO flat board, it is good that it is scoring to the situation of growing on the QDO flat board, is scoring to become blueness (as shown in Figure 7) on QDO-X-α-gal flat board.
2.7 library screening
Yeast mating liquid is applied on the QDO flat board, 30 ℃ be inverted to cultivate the bacterium colony on the picking flat board behind about 7d (＞2mm) to new QDO-X-α-Gal flat board, continue to cultivate, with the positive control is reference, to become blue bacterium colony renewed vaccination behind the cultivation 36hr and in the dull and stereotyped grid of new QDO-X-α-Gal, carry out blue hickie screening, obtain 23 blue clones (as shown in Figure 8) altogether.
The mating test of (2.8Y187 AD carrier library) and AH109 (pGBKT7-VP60)
Y187 (AD carrier library) and each picking one mono-clonal of AH109 (pGBKT7-VP60), be seeded among 0.5mL2 * YPDA, 23 altogether, get 100 μ L and be seeded to QDO/X-α-Gal flat board after with 0.5 * YPDA behind 30 ℃ of 50r/min * 24hr by dilution in 1: 10, be inverted 30 ℃ of cultivations.After five days, in 23 flat boards the blue hickie that quantity does not wait appears.Show that taking turns 23 library plasmids that filter out at yeast two-hybrid system five is true positives library plasmid.
In this research,, from rabbit liver tissue cDNA library, angle and obtain interactional albumen with rabbit hemorrhagic disease virus structural protein VP60 by yeast-two hybrid technique.Find that through order-checking and sequence alignment analysis this albumen is 215M22 kappa1 light chain immunoglobulin (Ig), Genbank accession number AY495826.1.
Experimental example 2 215M22 kappa1 segmental clone of proteic main encoding gene and expression
Design a pair of primer, introduce restriction enzyme site, upstream respectively: 5 ' CG
A TGG CCT ATG TCC TGT CTA C (EcoR I) 3 ', downstream: 5 ' CCG
CTA GAT GTC AGG CCA GAA AT (Xho I) 3 ' is a template with rabbit hepatic tissue cDNA, the main encode fragment of pcr amplification rabbit immunoglobulin 215M22 kappa1 light chain gene, about 480bp (as shown in Figure 9).Sequential analysis shows that this fragment is between encoding sequence 4546nt～5025nt, and sequence is shown in SEQ:NO.2.
Cut PCR product and expression vector pET32a with restriction enzyme EcoR I and Xho I enzyme, reclaim the purpose fragment.Handle through the T4DNA ligase enzyme, kappa1 light chain gene fragment is linked on the expression vector pET32a, obtain recombinant plasmid pKAPPA-1.Cut evaluation through restriction endonuclease EcoRI and Xho I enzyme once more, expression plasmid pKAPPA-1 successfully constructs, and enzyme is cut qualification result as shown in figure 10.
Expression plasmid pKAPPA-1 transformed into escherichia coli BL21 (DE3), this bacterial classification (BL21 (DE3) pKAPPA-1) is colon bacillus (Escherichia coli), be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, the address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, culture presevation is numbered: CGMCC No.4680, the culture presevation time is: on March 16th, 2011.Picking colony, inoculation LB substratum (kalamycin resistance) is cultivated, and after IPTG induces, gives expression to target protein kappa1 through evaluation, the about 17kD of this albumen (as shown in figure 11), the long 159aa of encoding amino acid sequence, sequence is shown in SEQ:NO.1.
Experimental example 3 215M22kappa1 albumen and the external checking of virus particle bonded
Purifying RHDV virus has obtained highly purified virus (shown in Figure 12) through electron microscopic observation, gel electrophoresis.
External co-immunoprecipitation test detects the interaction of reorganization 215M22 kappa1 albumen and virus particle.As capture antibody, catch virus particle and reorganization 215M22 kappa1 albumen composition with the rabbit hemorrhagic disease virus polyclonal antibody, detect specific signal with reorganization 215M22kappa1 protein antibodies.
KAPPA1 protein immunization co-precipitation (CO-IP) test:
1, mixing ProteinA/G-Agarose gently gets 40 μ L to the EP pipe of precooling, and 200 μ L cold crackings are separated Buffer washing balance Agarose beads, place on ice;
2,400 μ L cold crackings are separated Buffer and are diluted 14 μ L VP60 monoclonal antibodies, add in the EP pipe, and room temperature, behind low-speed oscillation 1-2h on the vibrator, 4 ℃, the centrifugal 3min of 1000r/m collects supernatant and makes SDS-PAGE and analyze;
3, Agarose beads precipitation is washed 3 times supernatant discarded with 400 μ L cracking Buffer;
4, the RHDV that adds 20 μ L purifying in Agarose beads precipitation places that low-speed oscillation spends the night on 4 ℃ of vibrators, and next day, 4 ℃, the centrifugal 3min of 1000r/m collects supernatant and makes SDS-PAGE and analyze the same washing 3 times;
5, add the KAPPA1 albumen of 200 μ L purifying in Agarose beads precipitation, low-speed oscillation spends the night on 4 ℃ of vibrators, and next day, 4 ℃, the centrifugal 3min of 1000r/m collects supernatant and does SDS-PAGE analysis, the same washing 3 times;
6, with the resuspended Agarose beads precipitation of 100 μ L, 1 * SDS loading buffer, boil 10min ,-20 ℃ of preservations;
7, get SDS-PAGE sample in 6 steps, carry out SDS-PAGE through 12% albumin glue, make Western Blotting and analyze, the positive contrast of KAPPA1 albumen of purifying is the negative contrast of Agarose beads in conjunction with RHDV.
(pvdf membrane is through the sealing of spending the night of 4 ℃ of 5% skimming milks, and next day, TBST washes 3 times, each 5min is hatched with the His-Tag mouse monoclonal antibody, 37 ℃ of effect 1h, TBST washes 3 times, each 5min, the sheep anti-mouse igg two that adds the HRP mark is anti-, hatches 1h for 37 ℃, TBST washes 3 times, each 5min, with the colour developing of DAB colour developing liquid, observations is (shown in Figure 13: 1, protein marker at last; 2, CO-IP result; 3, negative control; 4, positive control).
The above only is the preferred embodiments of the present invention, only is illustrative for the purpose of the present invention, and nonrestrictive; Those of ordinary skills understand, and can carry out many changes to it in the spirit and scope that claim of the present invention limited, revise, even the equivalence change, but all will fall within the scope of protection of the present invention.
Claims (10)
1. an albumen that combines with rabbit hemorrhagic disease virus VP 60 albumen is characterized in that described albumen comprises the sequence shown in SEQ ID NO:1 at least.
2. the albumen that combines with rabbit hemorrhagic disease virus VP 60 albumen as claimed in claim 1 is characterized in that described albumen is 215M22 kappa1 light chain immunoglobulin (Ig).
3. the albumen that combines with rabbit hemorrhagic disease virus VP 60 albumen as claimed in claim 1 is characterized in that described Argine Monohydrochloride sequence is shown in SEQ ID NO:1.
4. each described proteic nucleotide sequence of coding claim 1-3.
5. nucleotide sequence as claimed in claim 4 is characterized in that described nucleotide sequence is shown in SEQ ID NO:2.
6. host bacterium that contains the recombinant vectors of claim 4 or 5 described nucleotide sequences and contain this recombinant vectors.
7. a pharmaceutical composition for the treatment of rabbit haemorrhagic disease is characterized in that comprising each described albumen of claim 1-3 of effective dose and the carrier of pharmaceutically accepting.
8. pharmaceutical composition for the treatment of rabbit haemorrhagic disease is characterized in that comprising the claim 4 of effective dose or 5 described nucleotide sequences and the carrier of pharmaceutically accepting.
9. the application of each described albumen of claim 1-3 in preparation treatment rabbit haemorrhagic disease medicine.
10. claim 4 or 5 described nucleotides sequences are listed in the application in the preparation treatment rabbit haemorrhagic disease medicine.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104694482A (en) * | 2015-04-01 | 2015-06-10 | 中国农业科学院哈尔滨兽医研究所 | Hybridoma cell strain McAb 1G2 and rabbit hemorrhagic disease RHDV2 type virus monoclonal antibody |
| CN105175540A (en) * | 2015-10-15 | 2015-12-23 | 江苏省农业科学院 | Anti-rabbit hemorrhagic disease virus VP60 protein monoclonal antibody, B cell epitope recognized by same and application thereof |
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| WO2005007696A2 (en) * | 2003-07-15 | 2005-01-27 | Therapeutic Human Polyclonals, Inc. | Humanized immunoglobulin loci |
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| WO2005007696A2 (en) * | 2003-07-15 | 2005-01-27 | Therapeutic Human Polyclonals, Inc. | Humanized immunoglobulin loci |
| US7585668B2 (en) * | 2003-07-15 | 2009-09-08 | Therapeutic Human Polyclonals, Inc. | Humanized immunoglobulin loci |
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| F.ROS ET AL.: "Sequence analysis of 0.4 megabases of the rabbit germline immunoglobulin kappa1 light chain locus", 《ANIMAL GENETICS》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104694482A (en) * | 2015-04-01 | 2015-06-10 | 中国农业科学院哈尔滨兽医研究所 | Hybridoma cell strain McAb 1G2 and rabbit hemorrhagic disease RHDV2 type virus monoclonal antibody |
| CN105175540A (en) * | 2015-10-15 | 2015-12-23 | 江苏省农业科学院 | Anti-rabbit hemorrhagic disease virus VP60 protein monoclonal antibody, B cell epitope recognized by same and application thereof |
| CN105175540B (en) * | 2015-10-15 | 2018-11-23 | 江苏省农业科学院 | A kind of monoclonal antibody of anti-rabbit hemorrhagic disease virus VP 60 albumen and its B cell epitope and application of identification |
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