CN103570832A - Anti-Haemophilus parasuis porcine chimeric antibody, and construction method and application thereof - Google Patents
Anti-Haemophilus parasuis porcine chimeric antibody, and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses an anti-Haemophilus parasuis porcine chimeric antibody, and a construction method and application thereof. The porcine chimeric antibody is formed by connecting a light chain and a heavy chain through a disulfide bond and a non-covalent bond, wherein the light chain sequentially comprises a murine anti-Haemophilus parasuis monoclonal antibody light chain variable region and a porcine IgG light chain constant region from N end to C end, and the heavy chain sequentially comprises a murine anti-Haemophilus parasuis monoclonal antibody heavy chain variable region and a porcine IgG heavy chain constant region. The experiment proves that the anti-Haemophilus parasuis porcine chimeric antibody has obvious antibacterial effect in vivo and in vitro, and therefore, has wide application prospects in detecting or preventing Haemophilus parasuis infection.
Description
Technical field
The present invention relates to a boar source chimeric antibody and construction process and application, particularly a kind of pig source chimeric antibody and construction process and application that can resist haemophilus parasuis, belongs to biological medicine technology field.
Background technology
Haemophilus parasuis (Haemophilus parasuis, HPS) is non-haemolysis, the dependent Gram-negative bacteria of NAD, belongs to p pestic section.Haemophilus parasuis is a kind of important upper respiratory tract cause of disease bacterium of pig, is the cause of disease of Ge Laseshi sick (Gl sser ' s Disease).It is principal character that this disease be take meningitis, sacroiliitis and fiber disposition polyserositis, occurs sometimes septicemia and acute pneumonia symptom.In recent years, along with the change of swinery breeding way, as the application of early weaning scheme and 3 cultivating systems, the M & M of the swinery causing because of haemophilus parasuis increases year by year, and become one of the most serious bacteriosis in harm commercialization pig farm, particularly SPF pig farm.Although it is sick effectively to treat with microbiotic the Ge Laseshi that causes because of HPS, about the resistance of HPS, there have been a lot of reports.Vaccine is the effective ways of control Ge Laseshi disease, but because HPS serotype is numerous, and the vaccine of single serotype can only protect swinery to avoid the attack of homotype HPS, and has had a lot of research reports about HPS vaccine cross protection ability is poor.Therefore the method for, finding new control Ge Laseshi disease is very important.
Mouse source anti-haemophilus parasuis (Haemophilus parasuis; HPS) monoclonal antibody 1D8 can react with 15 serotype haemophilus parasuises; and can partly protect mouse to avoid homology and the attack of allos haemophilus parasuis volume; refer to document: haemophilus parasuis has the screening of protection effect monoclonal antibody and the evaluation of immunogenic protein; Tian Huabin; the > > of the < < Chinese Academy of Agricultural Sciences, 2012.Yet directly applying mouse resource monoclonal antibody on other kind animals may have problems.May be not and Fc acceptor and/or complementary reaction from the antibody of different genera animal, this can cause the shortage of suitable downstream function and be removed fast in vivo.Thereby produced in order to overcome the defect that mouse resource monoclonal antibody applies in pig body the chimeric antibody that comprises He Shuyuan variable region, pig Fc region.Utilize recombinant DNA technology produce chimeric antibody for the treatment of human diseases and pre-tetrandra root by wide coverage.The report that produces mouse-pig chimeric antibody is very few.The report that only has the mouse-pig chimeric antibody building for pig breeding and dyspnoea syndrome virus and Porcine epidemic diarrhea virus monoclonal antibody, but two pieces of articles all not mentioned chimeric antibody resist in vivo the ability of virus attack.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of new detection that can be used for haemophilus parasuis infection and the method for prevention.For this reason, the present invention proposes a kind of pig source chimeric antibody of anti-haemophilus parasuis, the pig source chimeric antibody that experimental results show that a kind of anti-haemophilus parasuis of the present invention is still external in vivo all has a fungistatic effect significantly.
In order to achieve the above object, the present invention has adopted following technique means:
The pig source chimeric antibody of a kind of anti-haemophilus parasuis of the present invention, it is characterized in that described antibody is connect and formed by disulfide linkage and non covalent bond by a light chain and a heavy chain, described light chain holds C end to comprise successively anti-haemophilus parasuis monoclonal antibody variable region of light chain, mouse source and pig IgG constant region of light chain from N, and described heavy chain comprises anti-haemophilus parasuis monoclonal antibody variable region of heavy chain, mouse source and pig IgG CH successively.
In the present invention, preferably, the aminoacid sequence of described anti-haemophilus parasuis monoclonal antibody variable region of light chain, mouse source is as shown in SEQ ID NO.1, the aminoacid sequence of described pig IgG constant region of light chain is as shown in SEQ ID NO.2, the aminoacid sequence of described anti-haemophilus parasuis monoclonal antibody variable region of heavy chain, mouse source is as shown in SEQ ID NO.3, and the aminoacid sequence of described pig IgG CH is as shown in SEQ ID NO.4.
In the present invention, preferred, described light chain and the N of heavy chain end also comprise signal peptide sequence, and preferred, the aminoacid sequence of described light chain is as shown in SEQ ID NO.5, and the aminoacid sequence of described heavy chain is as shown in SEQ IDNO.6.
The invention allows for the nucleotide sequence of pig source chimeric antibody described in the above any one of coding.
In the present invention, preferably, the nucleotide sequence of coding anti-haemophilus parasuis monoclonal antibody variable region of light chain, mouse source is as shown in SEQ ID NO.7, the nucleotide sequence of coding pig IgG constant region of light chain is as shown in SEQ ID NO.8, the nucleotide sequence of coding anti-haemophilus parasuis monoclonal antibody variable region of heavy chain, mouse source is as shown in SEQ ID NO.9, and the nucleotide sequence of coding pig IgG CH is as shown in SEQ ID NO.10.
In the present invention, preferably, 5 ' end of the nucleotide sequence of coding light chain and heavy chain is also connected with respectively the nucleotide sequence of coded signal peptide sequence, preferred, the nucleotide sequence of coding light chain is as shown in SEQ ID NO.11, and the nucleotide sequence of encoding heavy chain is as shown in SEQ ID NO.12.
In the present invention, preferred, described signal peptide sequence is as shown in SEQ ID NO.13, and the nucleotide sequence of this signal peptide of encoding is as shown in SEQ ID NO.14.
Further, the invention allows for a kind of expression vector, it is characterized in that containing the nucleotide sequence described in above any one.
In the present invention, preferred, described expression vector is rhabdovirus expression vector, preferred, and described expression vector is to build and obtain on the basis of two-way expression vector pFast-Bac-Dual.
Further, the invention allows for a kind of method of expressing the pig source chimeric antibody of described anti-haemophilus parasuis, it is characterized in that comprising the following steps:
(1) build for expressing the rhabdovirus expression vector of the pig source chimeric antibody of anti-haemophilus parasuis, wherein encode the nucleotide sequence of pig source chimeric antibody light chain as shown in SEQ ID NO.11, and the nucleotide sequence of coding pig source chimeric antibody heavy chain is as shown in SEQ ID NO.12;
(2) rhabdovirus expression vector transfection insect cell structure being obtained the assembling of carrying out baculovirus;
(3) the baculovirus inoculation insect cell obtaining, when cytopathy reaches 80-90%, the cell culture of results baculovirus infection, purifying, obtains.
In the present invention, preferred described rhabdovirus expression vector is to build and obtain on the basis of two-way expression vector pFast-Bac-Dual, the nucleotide sequence of the nucleotide sequence of described coding pig source chimeric antibody light chain and coding pig source chimeric antibody heavy chain is positioned at respectively PPH promotor on pFast-Bac-Dual or the downstream of p10 promotor separately, and described insect cell is sf-900 II.
Finally, the pig source chimeric antibody that the invention allows for described anti-haemophilus parasuis detects or prevents haemophilus parasuis to infect the application in reagent or medicine in preparation.And
The application of described expression vector in the pig source chimeric antibody of the anti-haemophilus parasuis of preparation.
Experiment showed, the chimeric antibody albumen that adopts method of the present invention to prepare, can suppress in vitro haemophilus parasuis growth; Can mouse in Mice Body avoid the attack of the haemophilus parasuis of lethal dose.Therefore, illustrating that chimeric antibody albumen of the present invention is still external in vivo all has a fungistatic effect significantly.
Accompanying drawing explanation
Fig. 1 is the PCR qualification result of the light CH of monoclonal antibody weight chain variable region Yu Zhu source antibody;
M:DNA Marker DL2000; 1: variable region of light chain; 2: constant region of light chain; 3: variable region of heavy chain; 4: CH.
Fig. 2 is recombinant plasmid light chain and heavy chain PCR qualification result;
1,3: light chain pcr amplification product; 2,4: heavy chain pcr amplification product; 5: negative control; M:DNAMarker DL2000
Fig. 3 is the carrier collection of illustrative plates of two-way expression vector pFast-Bac-Dual;
Fig. 4 is that light chain is connected to pFast-Bac-Dual double digestion qualification result afterwards;
1: the double digestion of light chain is identified collection of illustrative plates; M:DNA Marker DL2000
Fig. 5 is the double digestion qualification result of weight chain that is connected with the pFast-Bac-Dual plasmid of weight chain;
1: the double digestion qualification result of heavy chain; 2: light chain double digestion qualification result; M:DNA Marker DL2000
Fig. 6 is the carrier collection of illustrative plates of two-way expression vector pFast-Bac-Dual-H+L;
Fig. 7 is that P2 virus connects insect cell after malicious 72h and the contrast picture of control group insect cell;
Fig. 8 is the SDS-PAGE result after baculovirus expression;
M: albumen Marker1: the expressing protein after purifying
Fig. 9 is that the Western Blot after baculovirus expression detects collection of illustrative plates;
M: albumen Marker1: the Western Blot collection of illustrative plates of the albumen after purifying
Figure 10 is the ELISA comparing result of recombinant protein and monoclonal antibody 1D8;
Figure 11 is the interior bacteriostatic test of the body of recombinant protein.
Embodiment
Below in conjunction with specific embodiment, further describe the present invention, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment are only exemplary, scope of the present invention are not formed to any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can the details of technical solution of the present invention and form be modified or be replaced, but these modifications and replacement all fall within the scope of protection of the present invention.
The amplification of embodiment 1 monoclonal antibody weight chain variable region sequence and the light CH of pig antibody
1) amplification of anti-haemophilus parasuis monoclonal antibody variable region of light chain, mouse source and weight chain variabl area sequence
Anti-haemophilus parasuis (Haemophilus parasuis; HPS) preparation of monoclonal antibody 1D8 and evaluation refer to document: haemophilus parasuis has the screening of protection effect monoclonal antibody and the evaluation of immunogenic protein; Tian Huabin; the > > of the < < Chinese Academy of Agricultural Sciences, 2012.By Scientia Agricultura Sinica, study Harbin veterinary institute pig infectious ward swine disease molecular diagnosis component from also preserving.
After identifying the hypotype of antibody, monoclonal antibody cell is carried out to the extraction (extracting in a small amount test kit according to the total RNA of Axygen) of total RNA, and carry out reverse transcription.With primer BacVL-F, BacVL-R and BacVH-F, the BacVH-R of design, each primer sequence is as shown in table 1 below, and mouse source anti-haemophilus parasuis monoclonal antibody light chain variable region sequence and monoclonal antibody weight chain variabl area sequence increase respectively.
Table 1
(1) get 200 μ L cell cultures, add the Buffer R-Ι of 400 μ L, with the syringe of 1ml, repeatedly mix 8～10 times, proceed in the centrifuge tube of 1.5ml without RNA enzyme.
(2) add 150 μ L Buffer R-II, whirlpool concussion 15～30s, 12000 * g, 4 ℃ of centrifugal 5min.
(3) get supernatant and join in the 1.5ml centrifuge tube without RNA enzyme, add 250 μ L Virahols, mix.
(4) by preparing pipe, be placed in 2ml centrifuge tube (providing in test kit), the mixed solution in transfer step (3) is to preparing in pipe, 6000 * g, 4 ℃ of centrifugal 1min.
(5) abandon filtrate, by preparing pipe, put and get back in 2ml centrifuge tube, prepare in pipe and add 500 μ LBufferW1A, 12000 * g, 4 ℃ of centrifugal 1min.
(6) abandon filtrate, by preparing pipe, put and get back in 2ml centrifuge tube, prepare in pipe and add 700 μ LBufferW2,12000 * g, 4 ℃ of centrifugal 1min.Same operational applications W2 washes one time again.
(7) abandon filtrate, by preparing pipe, put and get back in 2ml centrifuge tube, 12000 * g, 4 ℃ of centrifugal 1min.
(8) by preparing pipe, put into a clean 1.5ml centrifuge tube without RNA enzyme, value is prepared the Buffer TE that periosteum central authorities add 70 μ L.
(9) the standing 1min of room temperature, 12000 * g, 4 ℃ of centrifugal 1min, obtain RNA.
(10) place 2min on ice after putting into 70 ℃ of water-bath 5min after the RNA7 μ L of wash-out is mixed with Random primer 1 μ L on ice, then add 5 * M-MLV Buffer5 μ L, dNTP Mixture (10mM) 2 μ L, Recombinant RNasin Ribonuclease Inhibitor0.5 μ L, Nuclease-Free Water8 μ L, after 37 ℃ of water-bath 50min, 70 ℃ of 15min obtain cDNA.
The PCR reaction system of amplification weight chain variable region is 50 μ L, 10 * Ex Taq buffer5 μ L, dNTP Mixture4 μ L, Ex Taq0.5 μ L, cDNA5 μ L, BacVL-F and BacVL-R or BacVH-F and BacVH-R each 1 μ L, PCR water 33 μ L.PCR reaction conditions: 94 ℃ of 5min, 94 ℃ of 1min, 53 ℃ of 30s, 72 ℃ of 1min, totally 30 circulations, 72 degrees Celsius of 10min, 4 ℃ of termination reactions.
2) amplification of pig antibody light chain constant region and CH
Get the inguinal lymph nodes of fresh pig, after grinding separated lymphocyte, extract its total RNA, the step of extracting is similar to total RNA method of extracting in cell, while just extracting total RNA from animal tissues, need to first extract the lymphocyte in lymphoglandula, the Buffer R-Ι that directly adds 400 μ L after extracting, step is afterwards identical with cell total rna extracting method.With primer BacCL-F and BacCL-R, BacCH-F and the BacCH-R of design, each primer sequence is as shown in table 2 below, and pig antibody light chain constant region and CH increase respectively.
Table 2
(1) in 200 object copper mesh, with the syringe of 5ml, lymphoglandula is pulverized, with the DKW lymphocyte separation medium of 1ml rinse copper mesh several under, proceed in 15ml centrifuge tube.
(2) along centrifugal tube wall, add serum-free 1640,800 * g of 1ml, 20 ℃, 30min gently.(lifting speed of whizzer is turned down to third gear)
(3) sucking-off buffy coat, adds in 15ml centrifuge tube, then adds 1640,250 * g, the 10min of 10ml ' serum-free.
(4) abandon supernatant, add the resuspended lymphocyte of complete RPMI-1640.
The PCR reaction system of light CH of increasing is 50 μ L, 10 * Ex Taq buffer5 μ L, dNTP Mixture4 μ L, Ex Taq0.5 μ L, cDNA5 μ L, BacCL-F and BacCL-R or BacCH-F and BacCH-R each 1 μ L, PCR water 33 μ L.PCR reaction conditions: 94 ℃ of 5min, 94 ℃ of 1min, 56 ℃ (light chain) 30s/62.5 ℃ of (heavy chain) 1min, 72 ℃ of 90s, totally 30 circulations, 72 ℃ of 10min, 4 ℃ of termination reactions.
2.2.3 agarose gel electrophoresis detects
(1) accurately take 1.0g agarose and join in 1 * TAE electrophoretic buffer of 100, after heating and melting, after room temperature is cooled to certain temperature, add 5 μ L ethidium bromides (EB), after mixing, pour glue groove into, treat that it solidifies.
(2) get after the PCR product of 5 μ L and 1 μ L6 * Loading buffer mix and join among glue groove, after add Marker.
(3) switch on power, voltage 150V, electric current 200mA, runs about 20min.
(4) glue is taken out, place observations in gel imaging instrument.
2.2.4PCR product glue reclaims
(1) in ultraviolet rubber tapping instrument, cut off respectively the Agarose plug that contains Mab1D8 variable region of light chain, variable region of heavy chain and pig antibody light chain constant region, CH, it is cut little as far as possible, put it into respectively in the centrifuge tube of 1.5ml, weigh its weight, the weight that deducts blank pipe obtains agarose weight, and this weight is as a gel volume (100mg=100 μ L volume).
(2) add the Buffer DE-A of three gel volumes, after mixing, in 75 ℃ of heating, be interrupted and mix (every 2-3min) until gel piece melts (approximately 6-8min) completely.
(3) add the Buffer DE-B of 0.5 Buffer DE-A volume, mix.
(4) draw the mixed solution in step (3), transfer to DNA preparation pipe (being placed in 2ml centrifuge tube), 12000 * g, 1min.
(5) by preparing pipe, put and get back in 2ml centrifuge tube, add 500 μ LBufferW1,12000 * g, 1min, abandons filtrate (6) and puts and get back in 2ml centrifuge tube preparing pipe, adds 700 μ LBufferW2,12000 * g, 1min, abandons filtrate.With same method again with 700 μ LBufferW2 washings once.
(7) by preparing pipe, put and get back in 2ml centrifuge tube, 12000 * g, 1min.
(8) by preparing pipe, be placed in clean 1.5ml centrifuge tube, add 25 μ L Eluent Buffer preparing film central authorities, the standing 1min of room temperature, 12000 * g, 1min, wash-out obtains the DNA of purifying.
2.2.5 connect and transform and order-checking
1) making of DH5 α competent cell
(1) get one of commercial DH5 α competence, melt on ice, with choosing a small amount of bacterium liquid of collarium picking, coat on nonresistant LB substratum 37 ℃ of overnight incubation
(2) picking list bacterium colony is added in the aseptic centrifuge tube of 1.5ml, adds the non-resistant LB nutrient solution of 1ml, and 37 degrees Celsius of concussions are spent the night.
(3) nutrient solution of drawing 100 μ L joins among the fresh non-resistant LB of 50ml, and 37 ℃ of concussions are cultivated.
(4) the OD value that 3h measures nutritive medium is afterwards cultivated in concussion, works as OD
600=0.4～0.6(measures OD value every 15～20min) time collect bacterial cultures for the preparation of competent cell.
(5) bacterium liquid is transferred in two 50ml centrifuge tubes aseptic, the precooling of use frozen water, on ice precooling 10min.
(6) 4600 * g, 4 ℃ of centrifugal 10min, outwell supernatant liquor gently, and centrifuge tube is inverted to 1min, then to the CaCl of precooling that adds the 0.1M of 12.5ml in precipitation
2, place 5min on ice.
(7) 4600 * g, 4 ℃ of centrifugal 3min, outwell supernatant, then in precipitation, add the CaCl of the precooling of 12.5ml0.1M
2, place 30min on ice.
(8) 3800 * g, 4 ℃ of centrifugal 3min, abandon supernatant, then in precipitation, add the CaCl of the precooling of 2ml0.1M
2, re-suspended cell, packing, every pipe 100 μ L ,-70 ℃ are frozen standby or be directly used in conversion.CaCl2 formula for the 0.1M that transforms is as shown in table 3 below.
Table 3 is for the CaCl2 formula of the 0.1M that transforms
2) glue is reclaimed and obtains to such an extent that purify DNA is connected, transforms, checks order with pMD18-T carrier
(1) in 0.5ml centrifuge tube, add pMD18-T carrier 1 μ L, purify DNA 3 μ L, ddH
2o1 μ L, mixes.
(2) add the Solution Ι of 5 μ L, mix rear 16 ℃ of connections and spend the night.
(3) take out four of DH5 α competent cells, melt on ice.
(4) connection product 2.2.4 joint being obtained joins respectively among four pipe competent cells, places 30min on ice.
(5) 42 ℃ of standing 90s of water-bath, then place 2min on ice.
(6) competent cell is transferred in the SOC substratum of 800 μ L, 45min is cultivated in 37 ℃ of concussions.
(7) get 100 μ L and evenly coat in the LB solid culture ware containing ammonia benzyl resistance, 37 ℃ of overnight incubation.
(8) picking colony carries out PCR evaluation, and positive bacterium colony is sent to Hua Da gene sequencing.
(9) sequencing sequence is the positive sequence that the present invention clones.
2.2.6 clone light chain total length and heavy chain total length
Again by BacVL-F and BacVL-R, BacVH-F and BacVH-R, BacCL-F and BacCL-R or BacCH-F and the resulting positive sequence of BacCH-R clone the present invention, then by merging method amplification light chain total length and the heavy chain total length of PCR, respectively with BacVL-F and BacCL-R amplification light chain full length sequence, with BacVH-F and BacCH-R amplification heavy chain full length sequence.Application KOD high-fidelity enzyme carries out PCR.PCR reaction system is as shown in table 4 below.
Table 4PCR reaction system
PCR result application DNA gel electrophoresis needs to see order-checking off after connection conversion after being accredited as the positive, take and determines that the weight chain being obtained is the sequence of wanting required for the present invention.Because KOD enzyme is the flat end PCR enzyme of a high-fidelity, so need to first add A when connecting, connect again afterwards conversion.
PCR product adds A application and adds A test kit and carry out, and it is as shown in table 3 below that PCR product adds A reaction system.
(1) in Eppendorf tube, add following adding " A " reaction solution, cumulative volume is 20 μ L
Table 5PCR product adds A reaction system
(2) 72 ℃ of reaction 20min.
(3) 72 ℃ of reaction 20min.
(4) place 1～2min on ice.
(5) by adding the DNA fragmentation that A completes, be connected with pMD18-T carrier, 16 ℃, connection is spent the night.
(6) connection product is transformed in DH5 α competent cell, picking list bacterium colony PCR identifies.
(7) positive colony is sent to order-checking.
PCR reaction conditions: 94 ℃ of 5min, 94 ℃ of 1min, 56 ℃ of (light chain) 30s or 56 ℃ of (heavy chain) 1min, 72 ℃ (light chain) 1min/72 ℃ of (heavy chain) 100s, totally 30 circulations, 72 ℃ of 10min, 4 ℃ of termination reactions.
Order-checking is returned afterwards by software analysis sequences such as DNASTAR, select wherein correct sequence, the positive colony of correct sequence is carried out to enlarged culturing, extract its plasmid, with BamH Ι and EcoR Ι, Xho Ι and Nhe Ι, carry out respectively double digestion, with DNA glue gel electrophoresis, identify that enzyme cuts result afterwards.
(1) get the bacterium liquid of 2ml overnight incubation in LB substratum, the centrifugal 1min of 12000 * g, abandons most supernatant.
(2) add 250 μ L Buffer S1 suspension bacterial precipitations, suspend and need evenly, should not leave little bacterium piece.
(3) add 250 μ L Buffer S2, gentleness also spins upside down fully to mix for 4～6 times and makes the abundant cracking of thalline, until form bright solution, this step should be avoided acutely rocking, otherwise can cause the pollution of genomic dna, and this step should not be over five minutes.
(4) add 350 μ L Buffer S3, gentle mixing 6～8 times, the centrifugal 10min of 12000 * g of also spinning upside down fully.
Draw the centrifuged supernatant in previous step and transfer to prepare in pipe and (prepare the centrifuge tube that pipe is placed in 2ml), the centrifugal 1min of 12000 * g, abandons filtrate.
(5) by preparing pipe, put and get back in centrifuge tube, add the BufferW1 of 500 μ L, the centrifugal 1min of 12000 * g, abandons filtrate.
(6) by preparing pipe, put and get back in centrifuge tube, add the BufferW2 of 700 μ L, the centrifugal 1min of 12000 * g, abandons filtrate; With same method, with the BufferW2 of 700 μ L, wash again once again, abandon filtrate.
(7) by preparing pipe, put and get back in 2ml centrifuge tube, the centrifugal 1min of 12000 * g.
(8) will prepare during pipe transfers in new 1.5ml centrifuge tube, the Eluent Buffer that adds 60 μ L in the central authorities of preparing film, the standing 1min of room temperature, the centrifugal 1min of 12000 * g, Eluent Buffer herein should first be heated to 65 ℃ of left and right, can mention elution efficiency like this.By the plasmid DNA obtaining according to showing 6(light chain below) and table 7(heavy chain) system join in 1.5ml centrifuge tube.
System is put among 37 ℃ of water-baths to enzyme cuts and spends the night.
System is put in 37 ℃ of water-baths to enzyme cuts and spends the night.
Enzyme is cut to product and carry out the evaluation of DNA gel electrophoresis, after qualification result is positive, enzyme is cut to product and carry out glue recovery, be directly used in connection expression vector.
2.2.7 the structure of expression plasmid and order-checking
First, by two-way expression vector pFast-Bac-Dual, its carrier collection of illustrative plates as shown in Figure 3, carries out unidirectional double digestion, first a side that connects heavy chain is carried out to double digestion, and double digestion system is as shown in table 8 below:
The enzyme system of cutting is put in 37 ℃ of water-baths to enzyme cuts and spends the night.
Enzyme is cut to the product spending the night and carry out DNA gel electrophoretic analysis, after band is correct, with restriction endonuclease Xho Ι and Nhe Ι, carry out double digestion, reclaim enzyme and cut product, the product after the product that glue is reclaimed reclaims with light chain double digestion glue is connected.Linked system is as shown in table 9 below.
16 ℃ of ligations are spent the night.
Connection product is transformed, be transformed in DH5 α competent cell, while transforming, when 37 ℃ of concussions are cultivated, 3h left and right is cultivated in concussion herein, then getting 100 μ L bacterium liquid evenly coats in the LB culture dish of ammonia benzyl resistance, 37 ℃ of overnight incubation, second day picking mono-clonal carries out PCR detection, and PCR is detected to positive mono-clonal enlarged culturing, extract plasmid and carry out double digestion evaluation, the clone of the qualification result positive send order-checking.
PCR reaction system: 94 ℃ of 5min, 94 ℃ of 1min, 56 ℃ of 1min, 72 ℃ of 1min totally 30 circulations, 72 ℃ of 10min, 4 ℃ of termination reactions.
Sequencing result feeds back afterwards according to software analysis sequences such as DNASTAR, find wherein identical with light chain masterplate of the present invention sequence, by clone's enlarged culturing of correct sequence, extract its plasmid, then carry out the double digestion of heavy chain direction, endonuclease reaction system is as shown in table 10 below:
37 ℃ of water-bath enzymes are cut and are spent the night.
Enzyme is cut to product and carry out the evaluation of DNA gel electrophoresis, be then connected with previous heavy chain double digestion product, the reaction system of connection is as shown in table 11 below:
16 ℃ of ligations are spent the night.
To connect product and transform DH5 α competent cell, the concussion incubation time of 37 ℃ of shaking tables that herein transform is also 3h left and right, gets afterwards the bacterium liquid of 100 μ L and evenly coats on the LB solid medium that contains ammonia benzyl resistance.37 ℃ of overnight incubation, second day picking mono-clonal carries out carrying out respectively the PCR evaluation of light chain and heavy chain and the evaluation of the double digestion of light chain and heavy chain after enlarged culturing.The mono-clonal bacterium liquid that identification reaction is positive is sent to Hua Da gene sequencing.After sequencing result feedback, applied biology software carries out sequence alignment analysis, finds out the positive colony of wanting required for the present invention, extracts its plasmid, be our the constructed two-way expression plasmid pFast-Bac-Dual-H+L for expressing, vector construction figure as shown in Figure 6.
2.2.8 the conversion of recombinant plasmid and extraction
1) conversion of recombinant plasmid
After the correct mono-clonal of order-checking is carried out to enlarged culturing, extract its plasmid, transform DH10Bac competent cell.The preparation of DH10Bac competent cell:
(1) on the dual anti-property LB flat board that contains 50 those resistances of μ g/ml card and 7 μ g/ml gentamicin resistances, use DH10Bac bacterium liquid drawing board, 37 ℃ of overnight incubation.
(2) single bacterium colony of picking from flat board, is inoculated in a dual anti-property liquid LB substratum that contains 50 those resistances of μ g/ml card and 7 μ g/ml gentamicin resistances, and in 37 ℃, 250rpm cultivates 3～6h, to OD
600it is 0.4～0.6 left and right.
(3) DH10Bac inoculum is hatched to 10min on ice, make culture be cooled to 0 ℃, proceed in the 50ml centrifuge tube of aseptic prior precooling, then in 4 ℃, centrifugal 8 minutes collecting cells of 5000rpm.
(4) pour out nutrient solution, supernatant is abandoned to the greatest extent as far as possible, then according to every 50ml initial incubation thing, add 30ml ice-cold 0.1CaCl in advance
2solution, suspension cell, places 30min on ice gently.
(5) in 4 ℃ of centrifugal 8min collecting cells of 5000rpm, abandon most supernatant.Add 4ml ice-cold 0.1M CaCl in advance
2.Suspension cell, adds 1ml through 80% glycerine of autoclaved prior precooling gently, and piping and druming mixes.
(6) by DH10Bac competent cell according to every pipe 100 μ L packing.Can directly with the DH10Bac competent cell of fresh preparation, carry out plasmid conversion, also can be placed on-70 ℃ of Refrigerator stores standby.
(7) get the correct plasmid 2 μ L of order-checking and join among the DH10Bac competent cell of 100 μ L, place 30min on ice.
(8) competent cell is put in 42 ℃ of water-baths and is hatched 90s, during do not rock competent cell.
(9) competent cell is taken out, place 2min on ice, the aseptic SOC nutrient solution that adds 800 μ L then, 37 ℃, 3h is cultivated in 250rpm concussion.
(10) getting 100 μ L bacterium liquid evenly coats on the three anti-LB solid mediums that contain 50 those resistances of μ g/ml card, 7 μ g/ml gentamicin resistances and 10 μ g/ml tetracyclines, be coated with the bacterium 30min elder generation X-gal of 40 μ L100 μ g/ml and IPTG of 16 μ L40 μ g/ml on three anti-substratum before, be used for carrying out blue hickie screening (because recombination efficiency is very low, so must apply the handsome choosing of blue hickie).
(11) picking white mono-clonal, continue to be inoculated on three anti-substratum of another new handsome choosing of blue hickie, observe the colony colour on second substratum, if the overwhelming majority or all white are positive, if contain most blue colonies, for false positive, need again to identify.
(12) the white mono-clonal on the substratum of picking programmed screening carries out enlarged culturing, extracts plasmid.Also blue negative clone of picking is carried out enlarged culturing simultaneously, for the negative control of later protein expression.
2) extraction of recombinant plasmid
The extraction of DH10Bac competent cell plasmid can not be with common plasmid extraction kit, measure very less, the plasmid extraction method that provides on the Bac-to-Bac Baculovirus Expression System handbook that provide according to Invitrogen company is extracted.
(1) with 10 aseptic μ L rifle choicests, be taken at the positive monoclonal bacterium colony of ruling on second three anti-substratum, join in a sterile test tube that contains 5ml tri-anti-liquid nutrient mediums.
24h is cultivated in (2) 37 ℃ of concussions.
(3) bacterium liquid is transferred in the aseptic centrifuge tube of 1.5ml, trim is centrifugal, 14000g * and, 1min, abandons most supernatant.
(4) the resuspended thalline of Solution Ι of use 0.3ml, the western thalline of blowing down on use rifle head gently, avoids violent piping and druming.
(5) add the Solution Ι Ι of 0.3ml and mix gently incubated at room 5min.
(6) slowly add 0.3ml3M Potassium ethanoate, when dripping, mix gently, muddy thing that at this moment will adularescent occurs, this is a linear protein matter and colibacillary genomic dna, places 5～10min on ice.
（7）14000g×，10min。
(8) supernatant liquor is transferred in a centrifuge tube that contains 0.8ml Virahol gently, notes not picking up any white depositions, mixes, and places 5～10min on ice.
(9) at ambient temperature 14000g *, 15min..
(10) gently discard supernatant liquor, note not picking up white depositions below, add the ethanol of 0.5ml70%, repeatedly clean white depositions several times.This step needs aseptic technique.
(11) 14000g *, 5min, repeating step 10,11 as required.
(12) the clean as far as possible supernatant that removes, does not carefully remove white solid below, aseptic dry 5～10min under room temperature condition, but too not dry.
(13) by the TE Buffer dissolved solids of 40 μ L, avoid violent purge,
(14) 4 ℃ of preservations.
The needed solution of plasmid DNA extracting in DH10Bac cell is as shown in table 12 below:
2.2.9 the transfection of insect cell and the assembling of baculovirus
Insect cell, before transfection, must guarantee that cell is in state of health, and in logarithmic phase, the quantitative requirement of cell is 1.5 * 10
6～2.5 * 10
6individual left and right.
(1) to the sf-900 II that adds 2ml in six orifice plates, then add wherein 8 * 10
5individual cell/ plate, then puts culture plate standing 15min at ambient temperature, makes cell attachment.
Method of counting: large lattice/4 * 16 * 10, cell count=4 in cumulative volume
4* extension rate
(2) mixed C ell fection II: the Cell fection II of 8 μ L is joined in the substratum of serum-free antibiotic-free of 100 μ L, whirlpool concussion 1～3s, more than this mixed solution will be placed 30min before using.
(3) recombinant plasmid of 1 μ g is added in the substratum of serum-free antibiotic-free of 100 μ L, mixes.
(4) mixed solution step 2 and 3 being obtained mixes, and room temperature is placed 30min.
(5) mixed solution in step 4 is added drop-wise on the Tissue Culture Plate of completing in step 1, cultivates 3～5h for 27 ℃.Remove transfection mixed solution, with the 1% dual anti-sf-900 II nutrient solution that contains of 2ml, cultivate.
In (6) 27 ℃ of incubators, cultivate until there is pathology.If the cytopathy of first-generation virus is not obvious, can after cell cultures 96h, collect containing virulent nutrient solution, transfer in the aseptic centrifuge tube of 15ml, centrifugal, 500 * g, 5min.
(7) supernatant liquor is transferred in new 15ml centrifuge tube, Here it is P1 is for virus, and 4 ℃ keep in Dark Place.
(8) with P1, repeat the 9th step operation after for virus inoculation insect cell, produce P2 for virus.
(9) then with P2, for virus, connect after insect cell and repeat the 9th step operation, produce P3 for virus.P3 is exactly the baculovirus of the restructuring of wanting required for the present invention for virus, can produce albumen for inoculation sf9 cell, and can produce obvious cytopathy.
2.2.10 protein purification
(1) get cell culture and cell conditioned medium in a small amount, carry out whether expressing protein of SDS configuration verification, where protein is present in, and belongs to soluble proteins or exists with inclusion body form.
(2) determine that the form that albumen is deposited with inclusion body exists.
(3) cell culture of results baculovirus infection, the centrifugal 10min of 10000rpm, abandons most supernatant, with PBS, washes three times, and the centrifugal 10min of 10000rpm, abandons most supernatant, weighs: test tube net weight M1, test tube weight in wet base M2, cell precipitation thing m=M2-M1.
(4) to NP-40 lysate (volume that adds lysate is to calculate with 10 times of ratios of cell mud volume) the suspension cell throw out that adds prior precooling in centrifuge tube, place 10min on ice, then 4 ℃, the centrifugal 10min of 10000rpm, abandons most supernatant.
(5) repeat to add NP40 lysate (volume is still 10 times of cell mud volume) the suspension cell throw out of prior precooling in centrifuge tube, and twice of supersound process, ultrasound condition: amplitude 28%, ultrasonic 5s, interval 10s, ultrasonic time 10min, getting the ultrasonic product of 10 μ L analyzes for SDS, then 4 ℃, the centrifugal 10min of 10000rpm, abandons most supernatant.
(6) to PBS damping fluid (adding volume is that 10 times of ratios of cell volume are calculated) the suspension cell throw out that adds prior precooling in centrifuge tube, and twice of supersound process: amplitude 28%, ultrasonic 5s, interval 10s, ultrasonic time 10min, gets the ultrasonic product of 10 μ L and analyzes for SDS, then 4 ℃, the centrifugal 10min of 10000rpm, abandons most supernatant.
(7) in test tube, add 2M urea (pH8.0) to wash precipitation, supersound process: amplitude 28%, ultrasonic 5s, interval 10s, ultrasonic time 10min, gets the ultrasonic product of 10 μ L and analyzes for SDS, and then 4 ℃, the centrifugal 10min of 10000rpm, abandons most supernatant.
(8) to the 8M urea dissolution precipitation thing that adds prior precooling in centrifuge tube.Then according to the step of Protein G protein purification post, carry out purifying.
(9) first with the Binding Buffer of 5～10ml, cross 0.45 μ m filter membrane aftertreatment purification column (cannot have air in purification column).
(10) then the sample of 2ml is crossed to 0.45 μ m membrane filtration in the aseptic penicillin bottle of 10ml, then got the Binding Buffer that 8ml crossed 0.45 μ m filter membrane and also join in penicillin bottle, with the pipettor of 1ml, repeatedly aspirate several times and mix.
(11) with the syringe of 1ml, sample was carried out to post, crossed in a new penicillin bottle, sample was crossed to post 3～5 times repeatedly, the slow post of crossing of trying one's best when crossing post, the post time that crosses of 1ml is greatly about 2min left and right.
(12) with the Binding Buffer of 5～10ml, cross 0.45 μ m filter membrane washing purification column (cannot have air in purification column).
(13) get 5ml Elution Buffer and cross 0.45 μ m filter membrane, then with the Elution Buffer that 1ml syringe is drawn 0.8ml, throw into fast in purification column and (make the pH in purification column from 7.0, drop to 2.7 rapidly), because liquid is now impure, give up.
(14) draw 1ml Elution Buffer and cross post with identical method, collect this liquid, then repeat this time to operate 3～4 times.
(15) with pH damping fluid, the albumen elutriant of collecting is carried out to pH regulator, by pH regulator to 7.0 left and right.
(16) directly use albumen or 4 ℃ of preservations.
(17) with the Elution Buffer of 5～10ml, wash purification column.
(18) with the Binding Buffer of 5～10ml, wash purification column again.
(19) finally use again 20% the washing with alcohol of 5～10ml, then pillar is put into 4 ℃ and saved backup.
The required various solution of inclusion body purification are as shown in table 13 below,
2.2.11ELISA detect protein-active
(1) with the coated elisa plate (package amount 2 μ g/ holes) of haemophilus parasuis strain isolated HLJ-018, hatch 1h for 37 ℃, 4 ℃ of coated spending the night.
(2) use PBST repetitive scrubbing elisa plate three times, each 3min.
(3) with 5% skim-milk, seal 37 ℃ of sealing 2h.
(4) outwell confining liquid, with PBST, wash elisa plate, repetitive scrubbing three times, each 3min.
(5) elisa plate is dried standby.
(6) to dry elisa plate in add the good albumen of purifying, 100 μ l/ holes, hatch 1h for 37 ℃.
(7) outwell supernatant liquor, PBST washing three times, adds goat-anti pig two anti-, hatches 1h for 37 ℃.
(8) outwell supernatant liquor, PBST washing three times, adds nitrite ion, colour developing 10min.
(9) enzyme mark detector is measured OD
450value.
2.2.12SDS-PAGE with Western blot analyzing proteins
1) SDS-PAGE analyzes
(1) SDS-PAGE solution formula, as shown in table 14 below:
Table 14
The solution preparing is joined and in gel slab, prepares respectively upper strata Jiao Yu lower floor glue.
(2) after the gelling admittedly of upper strata Jiao Yu lower floor, transfer in Vertial electrophorestic tank, add pretreated sample (in advance by the sample of 10 μ l and 8 μ l2 * Loading Buffer and the DTT of 2 μ l mix and boil 5min, rear centrifugal) and protein Marker.
(3) 80V voltage is run through spacer gel, make albumen in lower floor compared with beginning in same position.
(4) 120V voltage is run through separation gel.
(5) albumin glue of running through is carried out to coomassie brilliant blue staining, room temperature shaking table 2h, another piece albumin glue is analyzed for Western Blot.
(6) albumin glue of dyeing is put into decolouring box and added a certain amount of distilled water, microwave oven boils 5min, then outwells distilled water, the distilled water renewing, and, in repetitive operation 3～4 times till there is obvious band.
2) Western Blot analyzes
(1) when albumin glue will be run through, filter paper and cellulose nitrate film (NC film) are printed wet with transferring film liquid and distilled water respectively completely.
(2) glue and filter paper and NC film are placed successively according to the order of filter paper, NC film, protein adhesive, filter paper from top to bottom, then with scissors, according to the size of albumin glue, prune the size of filter paper and film, the edge of filter paper and film must be neat, puts into half-dried transferring film instrument and carry out transferring film after having pruned.
(3) time of transferring film, voltage: 15V/30min.
(4) NC film transferring film being completed is put into glass dish after taking out, and 5% 4 ℃ of skimming milks sealing is spent the night.
(5) outwell skimming milk, add PBST washing NC film, wash each 3min three times.
(6) to adding in vial plate albumen that purifying is good as primary antibodie, room temperature shaking table is hatched 2h.
(7) outwell protein solution, add PBST washing NC film, wash each 3min three times.
(8) add the anti-diluent of goat-anti pig two, room temperature shaking table is hatched 1h.
(9) outwell the anti-diluent of goat-anti pig two, add PBST washing NC film, wash each 3min three times.
(10) add the colour developing of HRP-DAB nitrite ion, observation experiment result.
2.2.13 the external neutralization test of albumen
(1) get frozen haemophilus parasuis HLJ-018, with choosing collarium, dip bacterium liquid, streak culture bacterium on TSA substratum, 37 ℃ of overnight incubation.
(2) picking mono-clonal bacterium colony, is inoculated in complete TSB liquid nutrient medium, and the concussion of 37 ℃ of shaking tables is cultivated, and when it is during in logarithmic phase, draws 1ml and makes its OD value be approximately 0.634 left and right as doubling dilution, and the bacterial number also having in it is approximately 0.24 * 10
3cFU/ml.
(3) put in 37 ℃ of water-baths after respectively the albumen of PBS, Mab1D8 and purifying being mixed with the bacterium liquid having diluted and hatch 1h.
(4) take out mixture, leave away in wink except the globule on tube wall, every pipe takes out 100 μ l and evenly coats on TSA substratum, 37 ℃ of overnight incubation.
(5) colony number on substratum is counted to contrast effect.
2.2.14 the neutralization test,in vivo of albumen
(1) get the female mouse of Balb/c in 18 6 week ages, be divided at random three groups, six every group, respectively as negative control group, positive controls and experimental group.
(2) 1h before attacking bacterium, the albumen of abdominal cavity passive immunization PBS, Mab1D8 and purifying, the then haemophilus parasuis (10 of the lethal dose of abdominal injection in logarithmic phase
10cFU/ml).
(3) observe the death condition of mouse, record experimental result.
2.3 test-results
2.3.1 the clone of the amplification of monoclonal antibody 1D8 weight chain variable region and antibody weight chain variable region, pig source
By the extraction of the total RNA of genome to monoclonal antibody 1D8 and pig lymphocyte, and be cDNA by its reverse transcription, the synthetic primer of application the present invention, carry out pcr amplification, 1% agarose gel electrophoresis, obtain the object band that size is about 336bp, 330bp, 345bp and 987bp, corresponding is monoclonal antibody variable region of light chain, mouse source, pig constant region of light chain, monoclonal antibody variable region of heavy chain, mouse source and pig CH, and result as shown in Figure 1.The nucleotide sequence of coding monoclonal antibody variable region of light chain, mouse source, pig constant region of light chain, monoclonal antibody variable region of heavy chain, mouse source and pig CH is respectively as shown in SEQ ID NO.7-10, wherein the N of mouse source monoclonal antibody variable region of light chain and monoclonal antibody variable region of heavy chain, mouse source end is connected with signal peptide sequence, as shown in SEQ ID NO.13.
2.3.2 the clone of recombinant plasmid light chain and heavy chain
Amplifying monoclonal antibody variable region of light chain, mouse source (LV), pig constant region of light chain (LC), monoclonal antibody variable region of heavy chain, mouse source (HV) and pig CH (HC) afterwards, by merging light chain and the heavy chain of the method amplification recombinant protein of PCR, through pcr amplification, 1% agarose gel electrophoresis, obtain two DNA bands that size is about 750bp and 1300bp, be respectively light chain and the heavy chain of recombinant protein, as Fig. 2, coding light chain and heavy chain nucleotide sequence respectively as shown in SEQ ID NO.11 and 12.
2.3.3 enzyme is cut evaluation recombinant plasmid
After light chain has connected pFast-Bac-Dual plasmid, with Xho Ι and Nhe Ι, carry out double digestion evaluation, obtain object band and a shuttle vector DNA of an about 750bp, as Fig. 4.
2.3.4 the double digestion qualification result of heavy chain
On being connected with the pFast-Bac-Dual plasmid of light chain by connecting heavy chain DNA sequence dna after BamH Ι and EcoR Ι double digestion, then by double digestion, identify heavy chain connection result, after BamH Ι and EcoR Ι double digestion, the object band and the plasmid DNA band that obtain a treaty 1500bp left and right, result is as Fig. 5.The carrier collection of illustrative plates of two-way expression vector pFast-Bac-Dual-H+L as shown in Figure 6.
2.3.5 baculovirus transfection insect cell
With transfection reagent by restructuring plasmid DNA transfection Sf9 cell, 27 ℃ cultivate five days after, there is no cytopathy, then the centrifugal 5min of 500 * g room temperature, continues inoculation insect cell with supernatant liquor, cultivates for 27 ℃ and within three days, has found slight cytopathy afterwards, then collect virus, the centrifugal 5min of 500 * g room temperature, continues inoculation insect cell with supernatant liquor, cultivates for 27 ℃ and within three days, has found obvious cytopathy afterwards.Result is as Fig. 7.
2.3.6 the SDS-PAGE after protein expression and Western Bolt detect
After the P3 of high-titer that obtains baculovirus is for virus, with it, inoculate insect cell, at postvaccinal the 3rd day after cytopathy reaches 80-90%, collecting cell, the centrifugal 5min of 500 * g room temperature, collect supernatant liquor and preserve as virus, precipitation is carried out purifying by inclusion body purification method, detects the expression of albumen by SDS-PAGE and Western Bolt.As Fig. 8 and 9:
2.3.7 the ELISA detected result of the albumen of baculovirus expression
Recombinant protein, through resulting albumen after protein purification, detects the activity of its protein by ELISA detection method, result as shown in figure 10.
2.3.8 the extracorporeal bacteria inhibitor test of the recombinant protein of baculovirus expression
After ℃ water-bath 30min of the haemophilus parasuis HLJ-01837 in vegetative period of the good albumen of purifying and separation and Culture, get 100 μ l bacterium liquid and evenly coat 37 ℃ of overnight incubation on TSA substratum, then the bacterium on substratum is counted, result is as shown in Table 15:
2.3.9 bacteriostatic test in the body of the recombinant protein of baculovirus expression
1h before the haemophilus parasuis HLF-018 of the passive inoculation lethal dose of Balb/c mouse, in the abdominal cavity of mouse, inject respectively Mab1D8, the recombinant protein after purifying and isopyknic PBS after the purifying of 0.5mg, the survival condition of observing mouse, result is as Figure 11.
As seen from the figure, the mouse of negative control group after the haemophilus parasuis of injection lethal dose during at 18h with regard to dead four, and the mouse of negative control group is completely dead when 32h, though and two groups of small mouses of recombinant protein group of the present invention and monoclonal antibody 1D8 have death, but ining contrast to negative control group profile albumen of the present invention has good bacteriostatic activity.
Claims (12)
1. the pig source chimeric antibody of an anti-haemophilus parasuis, it is characterized in that described antibody is connect and formed by disulfide linkage and non covalent bond by a light chain and a heavy chain, described light chain holds C end to comprise successively anti-haemophilus parasuis monoclonal antibody variable region of light chain, mouse source and pig IgG constant region of light chain from N, and described heavy chain comprises anti-haemophilus parasuis monoclonal antibody variable region of heavy chain, mouse source and pig IgG CH successively.
2. pig as claimed in claim 1 source chimeric antibody, it is characterized in that the aminoacid sequence of described anti-haemophilus parasuis monoclonal antibody variable region of light chain, mouse source is as shown in SEQ ID NO.1, the aminoacid sequence of described pig IgG constant region of light chain is as shown in SEQ ID NO.2, the aminoacid sequence of described anti-haemophilus parasuis monoclonal antibody variable region of heavy chain, mouse source is as shown in SEQ ID NO.3, and the aminoacid sequence of described pig IgG CH is as shown in SEQ ID NO.4.
3. pig as claimed in claim 1 or 2 source chimeric antibody, it is characterized in that also comprising signal peptide sequence at the N of described light chain and heavy chain end, preferably, the aminoacid sequence of described light chain is as shown in SEQ ID NO.5, and the aminoacid sequence of described heavy chain is as shown in SEQ ID NO.6.
4. the nucleotide sequence of pig source chimeric antibody described in the claim 1-3 any one of encoding.
5. nucleotide sequence as claimed in claim 4, it is characterized in that encoding the nucleotide sequence of anti-haemophilus parasuis monoclonal antibody variable region of light chain, mouse source as shown in SEQ ID NO.7, the nucleotide sequence of coding pig IgG constant region of light chain is as shown in SEQ ID NO.8, the nucleotide sequence of coding anti-haemophilus parasuis monoclonal antibody variable region of heavy chain, mouse source is as shown in SEQ ID NO.9, and the nucleotide sequence of coding pig IgG CH is as shown in SEQ ID NO.10.
6. nucleotide sequence as claimed in claim 4, it is characterized in that being also connected with respectively the nucleotide sequence of coded signal peptide sequence at 5 ' end of the nucleotide sequence of coding light chain and heavy chain, preferably, the nucleotide sequence of coding light chain is as shown in SEQ ID NO.11, and the nucleotide sequence of encoding heavy chain is as shown in SEQ ID NO.12.
7. an expression vector, is characterized in that containing the nucleotide sequence described in claim 4-6 any one.
8. expression vector as claimed in claim 7, is characterized in that described expression vector is rhabdovirus expression vector, and preferred, described expression vector is to build and obtain on the basis of two-way expression vector pFast-Bac-Dual.
9. express a method for the pig source chimeric antibody of anti-haemophilus parasuis claimed in claim 1, it is characterized in that comprising the following steps:
(1) build for expressing the rhabdovirus expression vector of the pig source chimeric antibody of anti-haemophilus parasuis, wherein encode the nucleotide sequence of pig source chimeric antibody light chain as shown in SEQ ID NO.11, and the nucleotide sequence of coding pig source chimeric antibody heavy chain is as shown in SEQ ID NO.12;
(2) rhabdovirus expression vector transfection insect cell structure being obtained the assembling of carrying out baculovirus;
(3) the baculovirus inoculation insect cell obtaining, when cytopathy reaches 80-90%, the cell culture of results baculovirus infection, purifying, obtains.
10. method as claimed in claim 7, it is characterized in that described rhabdovirus expression vector is to build and obtain on the basis of two-way expression vector pFast-Bac-Dual, the nucleotide sequence of the nucleotide sequence of described coding pig source chimeric antibody light chain and coding pig source chimeric antibody heavy chain is positioned at respectively PPH promotor on pFast-Bac-Dual or the downstream of p10 promotor separately, and described insect cell is sf-900 II.
The pig source chimeric antibody of the anti-haemophilus parasuis described in 11. claim 1-3 any one detects or prevents haemophilus parasuis to infect the application in reagent or medicine in preparation.
The application of expression vector described in 12. claims 5 or 6 in the pig source chimeric antibody of the anti-haemophilus parasuis of preparation.
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| CN110376388A (en) * | 2019-08-16 | 2019-10-25 | 南京农业大学 | A kind of haemophilus parasuis antibody detection method and its kit |
| CN112794915A (en) * | 2021-01-07 | 2021-05-14 | 重庆市畜牧科学院 | Anti-pig IgG monoclonal antibody and application thereof |
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| CN102876635A (en) * | 2012-09-28 | 2013-01-16 | 广东省农业科学院兽医研究所 | Haemophilus parasuis outer membrane protein P5 (OMP5) resistant monoclonal antibody, hybridoma cell strain and application |
| CN102876636A (en) * | 2012-09-28 | 2013-01-16 | 广东省农业科学院兽医研究所 | Monoclonal antibody of hemophilus parasuis resistant OMP5 (outer membrane protein), hybridoma cell strain HPS1E2 and application |
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| CN102876636A (en) * | 2012-09-28 | 2013-01-16 | 广东省农业科学院兽医研究所 | Monoclonal antibody of hemophilus parasuis resistant OMP5 (outer membrane protein), hybridoma cell strain HPS1E2 and application |
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| CN106749664A (en) * | 2016-11-24 | 2017-05-31 | 上海美迪西生物医药股份有限公司 | Preparation method of the restructuring EnbrelFab fragments in insect cell expression system |
| CN110376388A (en) * | 2019-08-16 | 2019-10-25 | 南京农业大学 | A kind of haemophilus parasuis antibody detection method and its kit |
| CN110376388B (en) * | 2019-08-16 | 2021-10-19 | 南京农业大学 | Haemophilus parasuis antibody detection method and kit thereof |
| CN112794915A (en) * | 2021-01-07 | 2021-05-14 | 重庆市畜牧科学院 | Anti-pig IgG monoclonal antibody and application thereof |
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