CN1522760A - Bivalent gene engineering vaccine of fowl infectious bursal disease and pasteurellosis - Google Patents
Bivalent gene engineering vaccine of fowl infectious bursal disease and pasteurellosis Download PDFInfo
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Abstract
The present invention discloses an avian infectious cloaced bursa disease and pasteurellosis bivalent gene engineering vaccine, relating to avian gene engineering vaccine. Said new vaccine is a pasteurella multocida C47-16/p GEX-KG-VP2, CCTCC No:M203056, on pGEX-KG plasmid is connected an infectious cloacal bursa VP2 protein gene, after induced expression said vaccine can be used for immuniza chicken by means of oral administration or injection mode to make chicken body interior produce antibody for resisting infectious cloacal bursa virus, and when a standard strong strain is used to challenge virus, it can provide protection action for chicken. At the same time said carrier bacterium self-body is a safe effective low viulent vaccine strain for preventing pasteurellosis, so that said vaccine strain also can provide protection for resisting avian pasteurellosis.
Description
Technical field
The present invention relates to fowl genetic engineering novel vaccine.Specifically, relate to poultry infectious bursal disease and pasteurellosis bivalent genetic engineering novel vaccine.
Background technology
Infectious bursal disease virus (IBDV) belongs to diplornavirus section, can cause chicken and turkey generation infectious bursal disease or Gumboro disease.It by the bone-marrow-derived lymphocyte that destroys chicken bursa cause 3～12 age in week chickling immunosuppressant.
The genome of IBDV is made up of 2 sections double-stranded RNAs.Less that section (A) coding VP1 is the RNA polymerase that double-stranded RNA relies on.That bigger section (B) contains 2 open frames of reading.Frame 1 is the VP5 encoding histone.Frame 2 is polyprotein coding, and this polyprotein can be processed into 3 virus proteins automatically by proteolytic enzyme, that is: VP2, VP4, VP3.Wherein VP2 and VP3 are this viral primary structure protein.VP2 is the main host protective antigen of IBDV.It contains 3 the former determinants of monoclonal antibody that independently can induce comprehensive antibody at least.
Because primary disease does not still have good Therapeutic Method so far, and immunity inoculation improves the special resistance of chicken, is the best approach of control IBD always.At present the vaccine that chicken is carried out immunity mostly is live vaccine, can be divided into 3 kinds on weak malicious type, toxic type and strong malicious type according to its virulence.Weak malicious type vaccine is easily neutralized by maternal antibody, and strong malicious type vaccine then seldom uses by force because of virulence.What comparatively generally use now is the toxic type vaccine.No matter be that weak malicious type or toxic type vaccine its immunization power in carrying out the attenuation process just lose gradually.The vaccine immunity failure of now often seeing is exactly the quality instability owing to these attenuated live vaccines, and reason caused in the appearance of variant and highly virulent strain etc.In addition, also might be that attenuated vaccine returns strong poison again, cause strong poison to disseminate.
Along with to the deepening continuously of IBDV molecular biological characteristic research, understanding of its variation mechanism and virulence determiner is also being deepened constantly.Many scientists efforts be made so that with engineered method and improve and strengthen epidemic prevention work to IBDV.Recent years, the report of existing the following aspects aspect the structural protein that use recombinant technique expression IBDV: the 1) research of subunit vaccine report; 2) at the VP2 of expression in escherichia coli albumen; 3) at the VP2 of yeast expression in vivo recombiant protein; 4) nucleic acid vaccine of IBDV research; 5) express the proteic recombinant herpesvirus of turkeys of IBDV VP2 (HVT): vHVT001, vHVT002.
Summary of the invention
We have cloned VP2 gene in the superpower street strain of IBDV of Jing Zhou, Hubei morbidity by engineered method, have made up prokaryotic expression plasmid pGEX-KG-VP2, and successfully at Pasteurells multocida C
48-16(be called for short: P.multocida C
48-16) in realized expression.Expression product is a soluble protein in the cytosol, can directly induce the antibody of chicken generation at IBDV with this bacterium, and provide protective effect to chicken.This recombinant vaccine is with a wide range of applications.
P.multocida C
48-16It is a kind of fowl cholera bacterial strain of weak poison.To be Heilungkiang veterinary drug one factory cultivate after 190 generations the less-virulent strain of breeding by 40 generations of Embryo Gallus domesticus with pasteurella multocida virulent strain to this bacterial strain again by Cavia porcellus.It is a kind of live vaccine of effective prevention fowl pasteurellosis as safe as a house.Cultivated the live bacterial vaccines of the prevention fowl pasteurella multocida disease that successfully always is China's first-selection so far from 1972.By we discover that it is a more satisfactory recombinant vaccine live vector bacterium, except that can be applicable to infectious bursal disease, also can be applicable to the research preparation of the recombinant vaccine of other disease.
In addition, the VP2 gene of the strong street strain of IBDV that we clone is not found the identical Strain of sequence with it in gene database (GenBank), therefore can affirm that it is a kind of new variation strain.
Therefore, the objective of the invention is to overcome the shortcoming and defect that prior art exists, the bivalent genetic engineering vaccine of a kind of poultry infectious bursal disease and Bacillus pasteurii disease is provided.Specifically:
1) provides a kind of bivalent genetic engineering vaccine that can prevent poultry infectious bursal disease and Bacillus pasteurii disease;
2) provide a kind of P.multocida of using C
48-16Bacterial strain prepares the method for live vector recombinant vaccine;
3) provide a kind of new IBDV Strain VP2 gene order.
The object of the present invention is achieved like this:
1, the cloning process of IBDV VP2 gene
1) extraction of pathological material of disease RNA
Get the sick chicken bursa of IBD and organize about 1 gram of pathological material of disease, add liquid nitrogen and grind, carry out RNA with sangon UNIQ-10Trizol RNA extraction agent box and extract.
2) the VP2 gene of RT-PCR amplification IBDV
Design sleeve type PCR primer carries out reverse transcription and the PCR first time with the overcoat primer, carries out the amplification second time with after the PCR product dilutes 1000 times for the first time with interior cover primer.The amplified production electrophoresis result is seen Fig. 1.
3) make up the pGEX-KG-VP2 plasmid
Amplified production is carried out electrophoresis and use a small amount of glue recovery test kit of Shanghai China Shun biological engineering company limited production to reclaim.Gel is reclaimed product be connected on the pMD18-T plasmid vector, transformed into escherichia coli DH5 α.
Extract the transformant plasmid DNA.The plasmid DNA and the pGEX-KG plasmid that have reclaimed with restriction endonuclease Bam HI and Hind III enzyme action.Electrophoresis reclaims the pGEX KG plasmid of VP2 gene and fragmentation.The VP2 gene is linked to each other with fragmentation pGEX-KG.Finish the structure of pGEX-KG-VP2 plasmid.With this bacterium transformed into escherichia coli DH5 α, purifying DNA plasmid behind the cultivation escherichia coli.
2, P.multocida C
48-16The preparation of competent cell and conversion
P.multocida C
48-16The preparation of competent cell
1) picking fowl pasteurella multocida C
48-16Single bacterium colony is in the culture fluid of LB+2% blood plasma, and 37 ℃ of shaken cultivation are spent the night.
2) get the P.multocida C of saturated cultivation
48-16LB culture fluid 400 microlitres were added in 40 milliliters of LB culture fluid 37 ℃ of shaken cultivation about 3 hours, obviously floccule occurred to culture fluid, put cooled on ice.Change in 50 milliliters of centrifuge tubes the centrifugal collection bacterial sediment of 5000g * 10min over to.
3) with 0.1mol/L mops rinsing once.
4) add 4 milliliters and contain 50mmol/L CaCl
2The resuspended thalline of 0.1mol/L mops solution, placed on ice 20 hours.
5) next day, that bacterium liquid is at 5000g * 5min, centrifugal.Remove supernatant, with containing 50mmol/L CaCl
2The resuspended thalline of 0.1mol/Lmops, this is P.multocida C
48-16Competent cell.
P.multocida C
48-16The transformation experiment of bacterial strain
1) gets P.multocida C
48-16Competent cell 200 microlitres add pGEX-KG-VP2 1 microgram, and ice bath 3 hours adds LB culture fluid 400 microlitres, and back 28 ℃ of joltings 2 hours are cooled off in 42 ℃ of water-baths 2 minutes.
2), coat on the flat board of LB-AMP-2% blood plasma 37 ℃ of overnight incubation with resuscitation fluid 100 microlitres.Inferior day by day have a colony growth.
Identify the bacterium colony that contains genes of interest with PCR.
This bacterial strain called after Pasteurella multocida C
48-16/ pGEX-KG-VP2.
3, the novel variant VP2 of infectious bursal disease virus sequencing analysis result
This strain is to have very strong pathogenic highly virulent strain, and the aminoacid sequence of its cDNA sequence and deduction is:
atg?aca?aac?ctg?caa?gat?caa?acc?caa?cag?att?gtt?ccg?ttc?ata?cgg????48
Met?Thr?Asn?Leu?Gln?Asp?Gln?Thr?Gln?Gln?Ile?Val?Pro?Phe?Ile?Arg
1???????????????5???????????????????10??????????????????15
agc?ctt?ctg?atg?cca?aca?acc?gga?ccg?gcg?tcc?att?ccg?gac?gac?acc???????96
Ser?Leu?Leu?Met?Pro?Thr?Thr?Gly?Pro?Ala?Ser?Ile?Pro?Asp?Asp?Thr
20??????????????????25??????????????????30
cta?gag?aag?cac?act?ctc?agg?tca?gag?acc?tcg?acc?tac?aat?ttg?act??????144
Leu?Glu?Lys?His?Thr?Leu?Arg?Ser?Glu?Thr?Ser?Thr?Tyr?Asn?Leu?Thr
35??????????????????40??????????????????45
gtg?ggg?gac?aca?ggg?tca?ggg?cta?att?gtc?ttt?ttc?cct?ggt?ttc?cct??????192
Val?Gly?Asp?Thr?Gly?Ser?Gly?Leu?Ile?Val?Phe?Phe?Pro?Gly?Phe?Pro
50??????????????????55??????????????????60
ggc?tca?att?gtg?ggc?gct?cac?tac?aca?ctg?cag?agc?aat?ggg?aac?tac??????240
Gly?Ser?Ile?Val?Gly?Ala?His?Tyr?Thr?Leu?Gln?Ser?Asn?Gly?Asn?Tyr
65??????????????????70??????????????????75??????????????????80
aag?ttc?gat?cag?atg?ctc?ctg?act?gcc?cag?aac?cta?ccg?gcc?agc?tac??????288
Lys?Phe?Asp?Gln?Met?Leu?Leu?Thr?Ala?Gln?Asn?Leu?Pro?Ala?Ser?Tyr
85??????????????????90??????????????????95
aac?tac?tgc?agg?cta?gtg?agt?cgg?agt?ctc?aca?gtg?agg?tca?agc?aca??????336
Asn?Tyr?Cys?Arg?Leu?Val?Ser?Arg?Ser?Leu?Thr?Val?Arg?Ser?Ser?Thr
100?????????????????105?????????????????110
ctc?cct?ggt?ggc?gtt?tat?gca?cta?aat?ggc?acc?ata?aac?gcc?gtg?acc??????384
Leu?Pro?Gly?Gly?Val?Tyr?Ala?Leu?Asn?Gly?Thr?Ile?Asn?Ala?Val?Thr
115?????????????????120?????????????????125
ttc?caa?gga?agc?ctg?agt?gaa?ctg?aca?gat?gtt?agc?tac?aat?ggg?ttg??????432
Phe?Gln?Gly?Ser?Leu?Ser?Glu?Leu?Thr?Asp?Val?Ser?Tyr?Asn?Gly?Leu
130?????????????????135?????????????????140
atg?tct?gca?aca?gcc?aac?atc?aac?gac?aaa?att?ggg?aac?gtc?cta?gta??????480
Met?Ser?Ala?Thr?Ala?Asn?Ile?Asn?Asp?Lys?Ile?Gly?Asn?Val?Leu?Val
145?????????????????150?????????????????155?????????????????160
ggg?gag?ggg?gta?acc?gtc?ctc?agc?tta?ccc?aca?tca?tat?gat?ctt?ggg??????528
Gly?Glu?Gly?Val?Thr?Val?Leu?Ser?Leu?Pro?Thr?Ser?Tyr?Asp?Leu?Gly
165?????????????????170?????????????????175
tat?gtg?aga?ctc?ggt?gac?ccc?att?ccc?gct?ata?ggg?ctc?gac?cca?aaa??????576
Tyr?Val?Arg?Leu?Gly?Asp?Pro?Ile?Pro?Ala?Ile?Gly?Leu?Asp?Pro?Lys
180?????????????????185?????????????????190
atg?gta?gca?aca?tgt?gac?agc?agt?gac?agg?ccc?aga?gtc?tac?act?ata??????624
Met?Val?Ala?Thr?Cys?Asp?Ser?Ser?Asp?Arg?Pro?Arg?Val?Tyr?Thr?Ile
195?????????????????200?????????????????205
act?gca?gcc?gat?gat?tac?caa?ttc?tca?tca?cag?tac?caa?gca?ggt?ggg??????672
Thr?Ala?Ala?Asp?Asp?Tyr?Gln?Phe?Ser?Ser?Gln?Tyr?Gln?Ala?Gly?Gly
210?????????????????215?????????????????220
gta?aca?atc?aca?ctg?ttc?tca?gct?aat?atc?gat?gcc?atc?aca?agc?ctc??????720
Val?Thr?Ile?Thr?Leu?Phe?Ser?Ala?Asn?Ile?Asp?Ala?Ile?Thr?Ser?Leu
225?????????????????230?????????????????235?????????????????240
agc?atc?ggg?gga?gaa?ctt?gtg?ttt?caa?aca?agc?gtc?caa?ggc?ctt?ata??????768
Ser?Ile?Gly?Gly?Glu?Leu?Val?Phe?Gln?Thr?Ser?Val?Gln?Gly?Leu?Ile
245?????????????????250?????????????????255
ctg?ggt?gct?acc?atc?tac?ctt?ata?ggc?ttt?gat?ggg?act?gcg?gta?atc??????816
Leu?Gly?Ala?Thr?Ile?Tyr?Leu?Ile?Gly?Phe?Asp?Gly?Thr?Ala?Val?Ile
260?????????????????265?????????????????270
acc?aga?gct?gtg?gcc?gcg?gac?aat?ggg?cta?acg?gcc?ggc?act?gac?aac??????864
Thr?Arg?Ala?Val?Ala?Ala?Asp?Asn?Gly?Leu?Thr?Ala?Gly?Thr?Asp?Asn
275?????????????????280?????????????????285
ctt?atg?cca?ttc?aat?att?gtg?att?cca?acc?agc?gag?ata?acc?cag?cca??????912
Leu?Met?Pro?Phe?Asn?Ile?Val?Ile?Pro?Thr?Ser?Glu?Ile?Thr?Gln?Pro
290?????????????????295?????????????????300
atc?aca?tcc?atc?aaa?ctg?gag?ata?gtg?acc?tcc?aaa?agt?ggt?ggt?cag??????960
Ile?Thr?Ser?Ile?Lys?Leu?Glu?Ile?Val?Thr?Ser?Lys?Ser?Gly?Gly?Gln
305?????????????????310?????????????????315?????????????????320
gcg?ggg?gat?cag?atg?tca?tgg?tca?gca?agt?ggg?agc?cta?gca?gtg?acg?????1008
Ala?Gly?Asp?Gln?Met?Ser?Trp?Ser?Ala?Ser?Gly?Ser?Leu?Ala?Val?Thr
325?????????????????330?????????????????335
atc?cac?ggt?ggc?aac?tat?cca?ggg?gcc?ctc?cgt?ccc?gtc?aca?cta?gta?????1056
Ile?His?Gly?Gly?Asn?Tyr?Pro?Gly?Ala?Leu?Arg?Pro?Val?Thr?Leu?Val
340?????????????????345?????????????????350
gcc?tac?gaa?aga?gtg?gca?aca?ggg?tct?gtc?gtt?acg?gtc?gcc?ggg?gtg?????1104
Ala?Tyr?Glu?Arg?Val?Ala?Thr?Gly?Ser?Val?Val?Thr?Val?Ala?Gly?Val
355?????????????????360?????????????????365
agc?aac?ttc?gag?ctg?atc?cca?aat?cct?gaa?cta?gca?aag?aac?ctg?gtc?????1152
Ser?Asn?Phe?Glu?Leu?Ile?Pro?Asn?Pro?Glu?Leu?Ala?Lys?Asn?Leu?Val
370?????????????????375?????????????????380
aca?gaa?tac?ggc?cga?ttt?gac?cca?ggg?gcc?atg?aac?tac?aca?aaa?ttg?????1200
Thr?Glu?Tyr?Gly?Arg?Phe?Asp?Pro?Gly?Ala?Met?Asn?Tyr?Thr?Lys?Leu
385?????????????????390?????????????????395?????????????????400
ata?ctg?agt?gag?agg?gac?cgt?ctt?ggc?atc?aag?acc?gta?tgg?cca?aca?????1248
Ile?Leu?Ser?Glu?Arg?Asp?Arg?Leu?Gly?Ile?Lys?Thr?Val?Trp?Pro?Thr
405?????????????????410?????????????????415
agg?gag?tac?act?gac?ttt?cgc?gag?tac?ttc?atg?gag?gtg?gcc?gac?ctc?????1296
Arg?Glu?Tyr?Thr?Asp?Phe?Arg?Glu?Tyr?Phe?Met?Glu?Val?Ala?Asp?Leu
420?????????????????425?????????????????430
aac?tct?ccc?ctg?aag?att?gca?gga?gca?ttt?ggc?ttc?aaa?gac?ata?atc?????1344
Asn?Ser?Pro?Leu?Lys?Ile?Ala?Gly?Ala?Phe?Gly?Phe?Lys?Asp?Ile?Ile
435?????????????????440?????????????????445
cgg?gcc?ata?agg?????????????????????????????????????????????????????1356
Arg?Ala?Ile?Arg
450
The IBDV sequence that sequence is the most close therewith in GenBank has:
gi|6519813|dbj|AB024076.1|Identities＝1347/1356(99％)
gi|9230678|gb|AF240686.1|AF240686?Identities＝1346/1356(99％)
gi|1669530|dbj|D49706.1|Identities＝1345/1356(99％)
gi|2262071|gb|L42284.1|IBDVKS?Identities＝1344/1356(99％)
gi|13957668|gb|AF262030.1|AF262030?Identities＝1340/1351(99％)
gi|28629198|gb|AF533670.1|Identities＝1343/1356(99％)
gi|1296812|emb|X92760.1|IBDVVP523?Identities＝1343/1356(99％)
gi|20805925|gb|AF508176.1|Identities＝1342/1356(98％)
4, Pasteurella multocida C
48-16The abduction delivering of/pGEX-KG-VP2
1) gets Pasteurella multocida C
48-16The single bacterium colony of/pGEX-KG-VP2 is inoculated in the LB-AMP culture fluid that contains 2% chicken serum.Cultivated 20 hours for 28 ℃.Thalline is reached capacity.
2) get 1% saturated bacterium liquid and transfer in 10 milliliters of LB-AMP culture fluid that contain 2% chicken serum, 28 ℃ of shaken cultivation add 400 microlitre 0.5mol/L lactose and cultivated 16 hours for 28 ℃ to obvious dregs occurring.
5, the antigenicity of abduction delivering product detects
1) culture fluid is changed in 10 milliliters of centrifuge tubes over to 5000g * 10 minute centrifugal collection thalline.Add 1 milliliter of resuspended thalline of TE.Add 50mg/ml lysozyme 20 microlitres, 1%Triton X-100,100 microlitres are put 37 ℃ of water-baths 1 hour.
2)-70 ℃ and room temperature freeze thawing 3 times.Ultrasonic disruption 10 times, each 30 seconds.
3) 4 ℃ of 1000g * 10 minute are got supernatant and are carried out the active detection of antigen protein.
A, agar immunodiffusion method detect expression product: the results are shown in Figure 2.
B, ELISA assay: use the positive antiserum of standard I BDV of Harbin Veterinary Medicine Inst., China Academy of Agriculture, expression product has been carried out the indirect ELISA detection, the results are shown in following table:
| Expression product content | 1/ 400 | ?1/ ?800 | ?1/ ?1600 | ?1/ ?3200 | ?1/ ?6400 | ?1/ ?12800 | ?1/ ?25600 | ?1/ ?51200 | ?0 | ?0 |
| Sample 1 OD value | 1.599 | ?1.590 | ?1.549 | ?0.916 | ?0.736 | ?0.562 | ?0.389 | ?0.315 | ?0.215 | ?0.235 |
| Sample 2 OD values | 1.587 | ?1.392 | ?1.261 | ?1.264 | ?0.889 | ?0.442 | ?0.310 | ?0.262 | ?0.182 | ?0.194 |
| Sample 3 OD values | 1.448 | ?1.254 | ?0.886 | ?0.767 | ?0.482 | ?0.297 | ?0.204 | ?0.192 | ?0.169 | ?0.167 |
OD value/positive criterion in blank OD value 〉=2.1 per sample, sample 1 is positive (P/N=2.50) when 1/12800 extension rate; Sample 2 is positive (P/N=2.35) when 1/12800 extension rate; Sample 3 is positive (P/N=2.87) when 1/6400 extension rate.
6, counteracting toxic substances protection experiment
1) material
Test chicken, the local chicken of 1 age in days.The strong malicious BC6/85 strain of IBDV standard is available from China Veterinary Drugs Supervisory Inst..The IBD live vaccine, FATRO S.P.A produces, available from Jingzhou City's veterinary station.IBD agar diffusion immunoprecipitation test (AGP) diagnostic antigen and positive serum, the development of Harbin veterinary institute.
2) method
A, test chicken are raised to 9 ages in days, to every chicken blood sampling, detect IBDV antibody in the serum with AGP.Test chicken IBD fine jade expands antibody should be negative.40 chickens with healthy, no IBD fine jade expansion antibody are divided into 4 groups, 10 every group at random.The A group, the oral weak toadstool of pasteurella multocida that is loaded with IBDV VP2 recombiant plasmid, 200,000,000/only; The B group, the oral weak toadstool of pasteurella multocida that is loaded with the VP2 recombiant plasmid of subcutaneous injection, 5,000 ten thousand/only; The C group, oral IBD live vaccine; The D group is the blank group.Twice of 10 ages in days and each immunity of 20 ages in days.
B, blood sampling at the inboard blood-letting of wing, are moulded pipe with 0.5ml and are collected.Before strong malicious BC6/85 strain challenge trial chicken, to every chicken blood sampling once, separate out serum and be made for AGP with the IBDV standard.
When c, 30 ages in days, the Total Test chicken is infected the strong malicious BC6/85 strain of IBDV standard, oral, 0.1ml/ is (the strong malicious BC6/85 strain dilution of 0.1gIBDV standard is 10ml) only.The strong malicious BC6/85 strain of IBDV standard was attacked back 72 hours, cutd open Total Test chicken extremely, the ANOMALOUS VARIATIONS of observing fabricius bursa.
The judgement of d, immanoprotection action size is directly related foundation with the lesion degree of fabricius bursa, is the indirect correlation foundation with the AGP production of antibodies of IBDV.The ANOMALOUS VARIATIONS of fabricius bursa on color and luster, has in various degree yellow or red; In size, enlargement is in various degree arranged, capsule internal wrinkles ruffle edge becomes circle; The outer surface serous coat has gel-shaped or cheesy exudate down and in the blister cavities, or hemorrhagic inflammation.
.3) result
Cut open the variation of inspection fabricius bursa behind the Table I counteracting toxic substances
The slight pathological changes of the obvious pathological changes of group test chicken is normal
A % % % only
A???????10????????????????????1?????10????9?????90
B???????10????????????????????2?????20????8?????80
C???????10????????1?????10????2?????20????7?????70
D???????10????????5?????50????3?????30????2?????20
In the Table I, through the t check, A group and D group, difference is extremely remarkable between B group and the D group; Significant difference between C group and the D group.
The test of Table II IBD agar diffusion immunoprecipitation detects the result of AGP antibody
The negative blood sample of the positive blood sample of group test chicken blood sample
Part part % part %
A???????????10????????10????100???0?????0
B???????????10????????10????100???0?????0
C???????????10????????9?????90????1?????10
D???????????10????????0?????0?????10????100
The result shows in Table I and the Table II, gives the oral and subcutaneous injection of chicken with toadstool a little less than being loaded with the pasteurella multocida of IBDV VP2 recombiant plasmid, all can produce significant immanoprotection action, and can stimulate the immune system of chicken to produce AGP antibody.
The present invention has the following advantages and good effect:
1, this genetic engineering bacterium can prevent infectious bursal disease and chicken Bacillus pasteurii disease, and epidemic prevention is effective, and safety is very good.
2, illustrated a kind of antigenic protein gene sequence of novel highly virulent strain, this sequence can be carried out the application and development of gene engineering antigen.
3, available this method is with Pasteurella multocida C
48-16Bacterial strain is developed to other recombinant vaccine.Because this bacterial strain China Application and Development three more than ten years, be considered to the live bacterial vaccines of low toxicity as safe as a house up till now always, production has the low advantage of production cost as vaccine development.
Description of drawings:
China typical culture collection center is used for the culture collection of proprietary program and accepts notice (receipt):
Classification name Pasteurella multocida C
48-16/ pGEX-KG-VP2
Preservation date on June 19th, 2003
Depositary institution China Wuhan Wuhan University postcode 430072
Deposit number CCTCC NO:M203056
Fig. 1 is an IBDV VP2 gene amplification electrophoretogram, wherein:
The colour developing of 1-amplification IBDV VP2 gene electrophoresis;
The colour developing of 2-200bp ladder electrophoresis;
Fig. 2 is P.multocida C
48-16/ pGEX-KG expression product agar immunodiffusion test figure, wherein:
The 3-IBDV fine jade expands positive serum (Harbin veterinary institute)
The undiluted expression product of 4-;
5-2 doubly dilutes expression product;
6-4 doubly dilutes expression product;
7-8 doubly dilutes expression product;
8-16 doubly dilutes expression product;
9-32 doubly dilutes expression product.
The specific embodiment
Application of the present invention has:
(1) is used for the prevention of infectious bursal disease and chicken Bacillus pasteurii disease.After should inducing with lactose by preceding method when being used for the prevention of infectious bursal disease, collect cell and wash several times the back and undertaken by water way or subcutaneous injection mode.Drinking-water dosage is equivalent to the saturated 1/3～1/2ml that induces bacterium liquid.Subcutaneous injection dosage is equivalent to the saturated 1/10～1/20ml that induces bacterium liquid.
(2) carry out other recombinant vaccine and gene engineering antigen research preparation with IBDV VP2 gene order of the present invention.
(3) available P.multocida C
48-16Bacterial strain is researched and developed as the live vector bacterium of other recombinant vaccine.
Claims (4)
1, a kind of poultry infectious bursal disease and pasteurellosis bivalent recombinant vaccine is characterized in that:
Pasteurella?multocida?C
48-16/pGEX-KG-VP2，CCTCC?NO：M203056；
On the pGEX-KG plasmid, be connected with infectious bursal disease VP2 protein gene; this bacterium is after carrying out abduction delivering; can make the antibody that produces the infectivity resistant bursal disease poison in the chicken body by oral or injecting immune chicken, with standard virulent strain counteracting toxic substances the time, provide protective effect for chicken.
2, the VP2 gene order of the novel street strain of a kind of infectious bursa of Fabricius virus is characterized in that its DNA sequence and deduced amino acid:
atg?aca?aac?ctg?caa?gat?caa?acc?caa?cag?att?gtt?ccg?ttc?ata?cgg????48
Met?Thr?Asn?Leu?Gln?Asp?Gln?Thr?Gln?Gln?Ile?Val?Pro?Phe?Ile?Arg
1???????????????5???????????????????10??????????????????15
agc?ctt?ctg?atg?cca?aca?acc?gga?ccg?gcg?tcc?att?ccg?gac?gac?acc????96
Ser?Leu?Leu?Met?Pro?Thr?Thr?Gly?Pro?Ala?Ser?Ile?Pro?Asp?Asp?Thr
20??????????????????25??????????????????30
cta?gag?aag?cac?act?ctc?agg?tca?gag?acc?tcg?acc?tac?aat?ttg?act????144
Leu?Glu?Lys?His?Thr?Leu?Arg?Ser?Glu?Thr?Ser?Thr?Tyr?Asn?Leu?Thr
35??????????????????40??????????????????45
gtg?ggg?gac?aca?ggg?tca?ggg?cta?att?gtc?ttt?ttc?cct?ggt?ttc?cct????192
Val?Gly?Asp?Thr?Gly?Ser?Gly?Leu?Ile?Val?Phe?Phe?Pro?Gly?Phe?Pro
50??????????????????55??????????????????60
ggc?tca?att?gtg?ggc?gct?cac?tac?aca?ctg?cag?agc?aat?ggg?aac?tac????240
Gly?Ser?Ile?Val?Gly?Ala?His?Tyr?Thr?Leu?Gln?Ser?Asn?Gly?Asn?Tyr
65??????????????????70??????????????????75??????????????????80
aag?ttc?gat?cag?atg?ctc?ctg?act?gcc?cag?aac?cta?ccg?gcc?agc?tac????288
Lys?Phe?Asp?Gln?Met?Leu?Leu?Thr?Ala?Gln?Asn?Leu?Pro?Ala?Ser?Tyr
85??????????????????90??????????????????95
aac?tac?tgc?agg?cta?gtg?agt?cgg?agt?ctc?aca?gtg?agg?tca?agc?aca????336
Asn?Tyr?Cys?Arg?Leu?Val?Ser?Arg?Ser?Leu?Thr?Val?Arg?Ser?Ser?Thr
100?????????????????105?????????????????110
ctc?cct?ggt?ggc?gtt?tat?gca?cta?aat?ggc?acc?ata?aac?gcc?gtg?acc????384
Leu?Pro?Gly?Gly?Val?Tyr?Ala?Leu?Asn?Gly?Thr?Ile?Asn?Ala?Val?Thr
115?????????????????120?????????????????125
ttc?caa?gga?agc?ctg?agt?gaa?ctg?aca?gat?gtt?agc?tac?aat?ggg?ttg????432
Phe?Gln?Gly?Ser?Leu?Ser?Glu?Leu?Thr?Asp?Val?Ser?Tyr?Asn?Gly?Leu
130?????????????????135?????????????????140
atg?tct?gca?aca?gcc?aac?atc?aac?gac?aaa?att?ggg?aac?gtc?cta?gta????480
Met?Ser?Ala?Thr?Ala?Asn?Ile?Asn?Asp?Lys?Ile?Gly?Asn?Val?Leu?Val
145?????????????????150?????????????????155?????????????????160
ggg?gag?ggg?gta?acc?gtc?ctc?agc?tta?ccc?aca?tca?tat?gat?ctt?ggg????528
Gly?Glu?Gly?Val?Thr?Val?Leu?Ser?Leu?Pro?Thr?Ser?Tyr?Asp?Leu?Gly
165?????????????????170?????????????????175
tat?gtg?aga?ctc?ggt?gac?ccc?att?ccc?gct?ata?ggg?ctc?gac?cca?aaa????576
Tyr?Val?Arg?Leu?Gly?Asp?Pro?Ile?Pro?Ala?Ile?Gly?Leu?Asp?Pro?Lys
180?????????????????185?????????????????190
atg?gta?gca?aca?tgt?gac?agc?agt?gac?agg?ccc?aga?gtc?tac?act?ata????624
Met?Val?Ala?Thr?Cys?Asp?Ser?Ser?Asp?Arg?Pro?Arg?Val?Tyr?Thr?Ile
195?????????????????200?????????????????205
act?gca?gcc?gat?gat?tac?caa?ttc?tca?tca?cag?tac?caa?gca?ggt?ggg????672
Thr?Ala?Ala?Asp?Asp?Tyr?Gln?Phe?Ser?Ser?Gln?Tyr?Gln?Ala?Gly?Gly
210?????????????????215?????????????????220
gta?aca?atc?aca?ctg?ttc?tca?gct?aat?atc?gat?gcc?atc?aca?agc?ctc????720
Val?Thr?Ile?Thr?Leu?Phe?Ser?Ala?Asn?Ile?Asp?Ala?Ile?Thr?Ser?Leu
225?????????????????230?????????????????235?????????????????240
agc?atc?ggg?gga?gaa?ctt?gtg?ttt?caa?aca?agc?gtc?caa?ggc?ctt?ata????768
Ser?Ile?Gly?Gly?Glu?Leu?Val?Phe?Gln?Thr?Ser?Val?Gln?Gly?Leu?Ile
245?????????????????250?????????????????255
ctg?ggt?gct?acc?atc?tac?ctt?ata?ggc?ttt?gat?ggg?act?gcg?gta?atc????816
Leu?Gly?Ala?Thr?Ile?Tyr?Leu?Ile?Gly?Phe?Asp?Gly?Thr?Ala?Val?Ile
260?????????????????265?????????????????270
acc?aga?gct?gtg?gcc?gcg?gac?aat?ggg?cta?acg?gcc?ggc?act?gac?aac????864
Thr?Arg?Ala?Val?Ala?Ala?Asp?Asn?Gly?Leu?Thr?Ala?Gly?Thr?Asp?Asn
275?????????????????280?????????????????285
ctt?atg?cca?ttc?aat?att?gtg?att?cca?acc?agc?gag?ata?acc?cag?cca????912
Leu?Met?Pro?Phe?Asn?Ile?Val?Ile?Pro?Thr?Ser?Glu?Ile?Thr?Gln?Pro
290?????????????????295?????????????????300
atc?aca?tcc?atc?aaa?ctg?gag?ata?gtg?acc?tcc?aaa?agt?ggt?ggt?cag????960
Ile?Thr?Ser?Ile?Lys?Leu?Glu?Ile?Val?Thr?Ser?Lys?Ser?Gly?Gly?Gln
305?????????????????310?????????????????315?????????????????320
gcg?ggg?gat?cag?atg?tca?tgg?tca?gca?agt?ggg?agc?cta?gca?gtg?acg????1008
Ala?Gly?Asp?Gln?Met?Ser?Trp?Ser?Ala?Ser?Gly?Ser?Leu?Ala?Val?Thr
325?????????????????330?????????????????335
atc?cac?ggt?ggc?aac?tat?cca?ggg?gcc?ctc?cgt?ccc?gtc?aca?cta?gta????1056
Ile?His?Gly?Gly?Asn?Tyr?Pro?Gly?Ala?Leu?Arg?Pro?Val?Thr?Leu?Val
340?????????????????345?????????????????350
gcc?tac?gaa?aga?gtg?gca?aca?ggg?tct?gtc?gtt?acg?gtc?gcc?ggg?gtg????1104
Ala?Tyr?Glu?Arg?Val?Ala?Thr?Gly?Ser?Val?Val?Thr?Val?Ala?Gly?Val
355?????????????????360?????????????????365
agc?aac?ttc?gag?ctg?atc?cca?aat?cct?gaa?cta?gca?aag?aac?ctg?gtc????1152
Ser?Asn?Phe?Glu?Leu?Ile?Pro?Asn?Pro?Glu?Leu?Ala?Lys?Asn?Leu?Val
370?????????????????375?????????????????380
aca?gaa?tac?ggc?cga?ttt?gac?cca?ggg?gcc?atg?aac?tac?aca?aaa?ttg????1200
Thr?Glu?Tyr?Gly?Arg?Phe?Asp?Pro?Gly?Ala?Met?Asn?Tyr?Thr?Lys?Leu
385?????????????????390?????????????????395?????????????????400
ata?ctg?agt?gag?agg?gac?cgt?ctt?ggc?atc?aag?acc?gta?tgg?cca?aca????1248
Ile?Leu?Ser?Glu?Arg?Asp?Arg?Leu?Gly?Ile?Lys?Thr?Val?Trp?Pro?Thr
405?????????????????410?????????????????415
agg?gag?tac?act?gac?ttt?cgc?gag?tac?ttc?atg?gag?gtg?gcc?gac?ctc????1296
Arg?Glu?Tyr?Thr?Asp?Phe?Arg?Glu?Tyr?Phe?Met?Glu?Val?Ala?Asp?Leu
420?????????????????425?????????????????430
aac?tct?ccc?ctg?aag?att?gca?gga?gca?ttt?ggc?ttc?aaa?gac?ata?atc????1344
Asn?Ser?Pro?Leu?Lys?Ile?Ala?Gly?Ala?Phe?Gly?Phe?Lys?Asp?Ile?Ile
435?????????????????440?????????????????445
cgg?gcc?ata?agg????????????????????????????????????????????????????1356
Arg?Ala?Ile?Arg
450
3, a kind of method for preparing poultry infectious bursal disease and pasteurellosis bivalent recombinant vaccine is characterized in that:
1) extraction of pathological material of disease RNA
Get the IBD chicken bursa of dying of illness and organize about 1 gram of pathological material of disease, add liquid nitrogen and grind, carry out RNA extraction with sangon UNIQ-10Trizol RNA extraction agent box;
2) the VP2 gene of RT-PCR amplification infectious bursa of Fabricius virus (IBDV)
Design sleeve type PCR primer carries out reverse transcription and the PCR first time with the overcoat primer, carries out the amplification second time with after the PCR product dilutes 1000 times for the first time with interior cover primer;
3) structure of pMD18-T-VP2 plasmid
Amplified production is carried out electrophoresis and use a small amount of glue recovery test kit of Shanghai China Shun biological engineering company limited production to reclaim; gel is reclaimed product be connected to and be configured to the pMD18-T-VP2 plasmid on the pMD18-T plasmid vector, with this plasmid transformation escherichia coli DH5 α.
4) make up the pGEX-KG-VP2 plasmid
The transformed bacteria that will have the bacillus coli DH 5 alpha of pMD18-T-VP2 plasmid is cultivated, and plasmid extracts, and with being connected with the pGEX-KG plasmid of same enzyme action behind the restriction endonuclease double digestion, obtains the pGEX-KG-VP2 plasmid.With connecting product transformed into escherichia coli DH5 α.Carry out the screening of transformant and the extraction purification of cultivation and pGEX-KG-VP2 plasmid then;
5) transform P.multocida C with the pGEX-KG-VP2 plasmid
48-16
P.multocida C
48-16The preparation of competent cell: picking P.multocida C
48-16Single bacterium colony is in the culture fluid of LB+2% blood plasma, and 37 ℃ of shaken cultivation are to saturated; Get culture fluid 400 microlitres, be added on that 37 ℃ of shaken cultivation were to cool off on the 0.1 rearmounted ice bath to culture fluid OD600 about 3 hours in 40 milliliters of LB culture fluid; The centrifugal collection thalline of 5000g * 10min.Contain 50mmol/L CaCl with adding 4 milliliters after the 0.1mol/L mops rinsing once
2The resuspended thalline of 0.1mol/L mops solution, placed on ice 20 hours.Bacterium liquid is centrifugal at 5000g * 5min.Remove supernatant, use 50mmol/L CaCl
2The resuspended thalline of 0.1mol/L mops, this is P.multocida C
48-16Competent cell;
P.multocida C
48-16Transformation experiment: get P.multocida C
48-16Competent cell 200 microlitres add the pGEX-KG-VP21 microgram, and ice bath 3 hours adds LB culture fluid 400 microlitres, and back 28 ℃ of joltings 2 hours are cooled off in 42 ℃ of water-baths 2 minutes.Resuscitation fluid 100 microlitres are coated on the flat board of LB-AMP-2% blood plasma 37 ℃ of overnight incubation; Inferior day by day have a colony growth;
Identify the strain that contains genes of interest with PCR; This strain called after Pasteurella multocida C
48-16/ pGEX-KG-VP2.
4, Psateurella multocida C
48-16The application of/pGEX-KG-VP2 is characterized in that:
1) is used for the prevention of infectious bursal disease and fowl pasteurellosis;
2) carry out other recombinant vaccine and gene engineering antigen research preparation with IBDV VP2 gene order of the present invention;
3) available P.multocida C
48-16Bacterial strain is researched and developed as the live vector bacterium of other recombinant vaccine.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007025431A1 (en) * | 2005-09-02 | 2007-03-08 | Zhigao Bu | Recombinant newcastle disease lasota attenuated vaccine strain expressing vp2 gene of infectious bursal disease virus |
| CN100360662C (en) * | 2004-09-27 | 2008-01-09 | 长江大学 | Preparation process and application of genetic engineering subunit vaccine of infectious bursal disease |
| CN100422329C (en) * | 2005-03-08 | 2008-10-01 | 浙江大学 | Method for preparing immortalized cell of protein for expressing gene code of infective buresa of Fabricius virus |
| CN102260649A (en) * | 2011-06-30 | 2011-11-30 | 河南农业大学禽病研究所 | Infectious bursal disease virus (IBDV) and method for preparing inactivated vaccines and combined vaccines by breeding IBDV with chick-embryo cell system and bioreactor |
| CN101704889B (en) * | 2009-11-13 | 2013-01-16 | 烟台绿叶动物保健品有限公司 | Method for preparing chicken bursal antibody |
| CN107475445A (en) * | 2017-08-16 | 2017-12-15 | 东北农业大学 | Differentiate kit and its application of chicken infectivity bursa of Fabricius virus highly-wetting liquid based on RT PCR and RFLP technology |
-
2003
- 2003-09-04 CN CNA03125375XA patent/CN1522760A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100360662C (en) * | 2004-09-27 | 2008-01-09 | 长江大学 | Preparation process and application of genetic engineering subunit vaccine of infectious bursal disease |
| CN100422329C (en) * | 2005-03-08 | 2008-10-01 | 浙江大学 | Method for preparing immortalized cell of protein for expressing gene code of infective buresa of Fabricius virus |
| WO2007025431A1 (en) * | 2005-09-02 | 2007-03-08 | Zhigao Bu | Recombinant newcastle disease lasota attenuated vaccine strain expressing vp2 gene of infectious bursal disease virus |
| CN101704889B (en) * | 2009-11-13 | 2013-01-16 | 烟台绿叶动物保健品有限公司 | Method for preparing chicken bursal antibody |
| CN102260649A (en) * | 2011-06-30 | 2011-11-30 | 河南农业大学禽病研究所 | Infectious bursal disease virus (IBDV) and method for preparing inactivated vaccines and combined vaccines by breeding IBDV with chick-embryo cell system and bioreactor |
| CN102260649B (en) * | 2011-06-30 | 2012-11-14 | 河南农业大学禽病研究所 | Infectious bursal disease virus (IBDV) and method for preparing inactivated vaccines and combined vaccines by breeding IBDV with chick-embryo cell system and bioreactor |
| CN107475445A (en) * | 2017-08-16 | 2017-12-15 | 东北农业大学 | Differentiate kit and its application of chicken infectivity bursa of Fabricius virus highly-wetting liquid based on RT PCR and RFLP technology |
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