CN101880314A - Bursin-like peptide of gene engineering as well as expression gene and application thereof - Google Patents
Bursin-like peptide of gene engineering as well as expression gene and application thereof Download PDFInfo
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Abstract
The invention relates to bursin-like peptide of gene engineering as well as an expression gene and application thereof, which belong to the technical field of the gene engineering. The bursin-like peptide of the gene engineering has an amino acid sequence shown as SEQ ID NO.1 and is coded by a gene fragment having a nucleotide sequence shown as SEQ ID NO.2. The gene fragment of the bursin-like peptide of the gene engineering, which is protected by the invention, can be accurately reproduced and expressed with high efficiency. After the gene is inserted into an expression gene pET-32a and is converted into a colibacillus BL21 (DE3), the content of bursin tripeptide (BS) can account for 28.6 percent of the total protein of a thallus by high-efficiency expression, and the requirement of the industrialized production is completely reached.
Description
Technical field
The present invention relates to the plain sample peptide of a kind of genetically engineered capsule and expressing gene and application, belong to gene engineering technology field.
Background technology
The plain tripeptides of capsule (BSTP or BS) is that a kind of sequence that Audhya T etc. purified from chicken bursa in 1986 first is Lys-His-Gly-NH
2Tripeptides, function is to induce bird and mammiferous B cell precursor to be divided into the B cell, selects medullarin, and humoral immunization is played important regulatory role.
Preparation method to the plain tripeptides of capsule mainly contains two kinds at present: a kind of is chemical synthesis, and another kind is a biological synthesis process.
Chemical process is because its intrinsic drawback, as: technology is loaded down with trivial details, and by product is many and remove difficulty etc.As publication number is the Chinese patent of CN1696151; disclose a kind of method for synthesizing bursa of Fabricius bursin, it comprises: amino acid protection, protection HIS-GLY methyl esters dipeptides are synthetic, the purifying of synthetic, the bursa of Fabricius bursin crude product of the deaminizating protection of synthetic, the protection Methionin-HIS-GLY of the deaminizating protection of protection HIS-GLY dipeptides, protection Methionin-HIS-GLY, bursa of Fabricius bursin crude product.
The synthetic main direction of biological method is to utilize the expressing gene importing intestinal bacteria of the plain tripeptides of capsule to carry out scale operation then.Because expression amount is low, Shang Weiyou can reach the report of industrial applications but so far.
Synthetic the highest being reported as of output: the Zhang Huanling etc. of biological method are called in the Jiangsu interim name of agricultural journal 2003-19-2: mentioned in the paper of the plain tripeptides Determination on content of capsule in the fabricius bursa extract success in intestinal bacteria carrying out the plain tripeptides of capsule express, the codon that it utilizes intestinal bacteria to habitually practise, 5 BSTP gene series connection are linked to each other with cloning vector pDuc57, at expression in escherichia coli, densitometric scan shows that BS content only accounts for 13.4% of bacterial protein.Li Ping etc. are called in the interim name of Chinese animal doctor's magazine 2006-42-4: mention the codon that has utilized intestinal bacteria to habitually practise in the gene clone of the plain tripeptides of capsule and the paper of expression, with synthetic BS1, BS2 is the plain tripeptides series connection of primer amplification capsule fragment (BS8), clone pBAD/Thio-TOPO carrier, express with the pBAD/TOPO-Thio escherichia expression system, the molecular weight size that contains the BS8 fusion rotein is about 19kDa, the BS8 fusion rotein mainly exists with soluble form, back extraction need be carried out cytoclasis, and the nickel post carries out purifying etc.But still exist the plain tripeptides expression amount of capsule not high, back extraction process needs steps such as cytoclasis, purifying, and cost can't carry out the drawback of scale operation than higher and loaded down with trivial details.
Therefore develop the high expression level amount, back extraction process cost is low, and step is simple, is fit to large-scale production, and the plain preparation method of highly active tripeptides capsule solves the plain tripeptides of present capsule to use one of main path of bottleneck.
Summary of the invention
At the deficiencies in the prior art, the invention provides the plain sample peptide of a kind of genetically engineered capsule and expressing gene and application.
Summary of the invention
Find after deliberation, increase for the BSTP gene at present and express the also fewer of report, BSTP can't scale operation, major cause is because the length of BSTP is less, only be three amino acid, the primer length when the design primer PCR is less like this, and annealing temperature is lower, the gene fragment accuracy that clones is lower and repeated relatively poor, and this had also just stayed very big difficulty for carrying out the BSTP gene clone afterwards and expressing.The present invention makes up pET-32a (+) expression vector according to designing a kind of BS sample peptide gene fragment according to the habitual codon of intestinal bacteria, and transformed into escherichia coli BL21 efficiently expresses in shaking bottle, adopts unconventional back extraction process to prepare the plain sample peptides products of capsule.
Detailed Description Of The Invention
The present invention relates to:
[1] the plain sample peptide of a kind of genetically engineered capsule has aminoacid sequence shown in SEQ ID N0.1.
The invention still further relates to:
[2] gene fragment of the plain sample peptide of genetically engineered capsule in a kind of coding [1] has nucleotide sequence shown in SEQID N0.2.
Further, the invention still further relates to:
[3] contain the expression vector of nucleotide sequence shown in the SEQID N0.2.
In addition, the invention still further relates to:
[4] application of the gene fragment of the plain sample peptide of genetically engineered capsule of basis [2] record in the plain tripeptides of preparation capsule.
[5] according to the application of [4] record, step is as follows:
(1) gene fragment of the plain sample peptide of genetically engineered capsule is inserted expression plasmid, obtain recombinant plasmid;
(2) the recombinant plasmid transformed host bacterium that step (1) is made makes up host bacterium expression system;
(3) the plain sample peptide of the host bacterium expression system abduction delivering capsule that utilizes step (2) to make up, and process aftertreatment technology makes the plain tripeptides of capsule.
[6] application of putting down in writing according to [5], described in the step (1) gene fragment of the plain sample peptide of genetically engineered capsule is inserted expression plasmid, be meant gene fragment with nucleic acid restriction endonuclease EcoR I and the plain sample peptide of one of SalI, Xho I or HindIII double digestion genetically engineered capsule, be connected on the expression plasmid that same enzyme cuts, carry out sequence verification then.
Above-mentioned nucleic acid restriction endonuclease EcoR I and SalI, Xho I or HindIII are prior art products, can buy by market.
[7] according to the application of [6] record, described expression plasmid is: pET-32a, pET-32b or pET-32c.Used expression plasmid is the commercially available prod, and those skilled in the art can operate according to product description, and the gene fragment of the plain sample peptide of genetically engineered capsule is inserted expression plasmid.
[8] according to the application of [5] record, the host bacterium is described in the step (2): e. coli bl21 (DE3).Above-mentioned host bacterium is the commercially available prod, and those skilled in the art can operate according to product description, with recombinant plasmid transformed host bacterium.
[9] according to the application of [5] record, the step of aftertreatment technology is as follows in the step (3):
The plain sample peptide of capsule after expressing bacterium liquid after centrifugal, is collected bacterial sediment,, mix ice bath 8～10min with 0 ℃ of ice-cold high-permeation solution suspension bacterial sediment; Then, centrifugal collection bacterial sediment with 0 ℃ of ice-cold hyposmosis solution suspension bacterial sediment of equal-volume, mixes ice bath 8～10min; Then, the centrifuging and taking supernatant liquor is crossed 0.2～0.24um cellulose mixture film, obtains the plain tripeptides of capsule.The plain sample peptide of abduction delivering capsule step can be operated in conjunction with this area ordinary method according to the products instruction of host bacterium.
[10] application of putting down in writing according to [9], described high-permeation solution: pH=8.5, final concentration Tris-HCl 20～30mmol/L, EDTA (ethylenediamine tetraacetic acid (EDTA)) 2～3mmol/L, sucrose 25wt%; Described hyposmosis solution is: pH=8.5, final concentration Tris-HCl 20～30mmol/L, EDTA 2～3mmol/L.
The used expression plasmid pET-32a of the present invention, escherichia coli DH5a, e. coli bl21 (DE3), TOP10 competent cell, 0.22um cellulose mixture film etc. are prior art products, can buy by market.
The gene fragment of the plain sample peptide of the genetically engineered capsule that the present invention protected; can duplicate expression accurately and efficiently; after this gene insertion expression vector pET-32a is transformed into e. coli bl21 (DE3); through efficiently expressing; BS (the plain tripeptides of capsule) content content can account for 28.6% of bacterial protein, reaches industrial production requirement fully.
Description of drawings
The plain sample peptide of Fig. 1 capsule gene SOE-PCR amplification electrophoresis picture; Wherein: swimming lane M:DL2000; Swimming lane 1,2, the 3:BS product;
The synoptic diagram of the plain sample peptide of Fig. 2 capsule expression vector pet32a-BS;
The SDS-PAGE electrophoresis picture of Fig. 3 target protein, wherein: swimming lane 1: standard molecular weight Marker; Swimming lane 2-3: inductive bacterium liquid not; Swimming lane 4,5,6: induce back pET-32a (+)-BS; Swimming lane 7,8: pET-32a (+)-BS behind the purifying.
Fig. 4 adopts Li Ping etc. at the interim electrophoresis picture of mentioning with the plain tripeptides of biological method synthetic capsular of Chinese animal doctor's magazine 2006-42-4, wherein: swimming lane 1: inductive bacterium liquid not; Swimming lane 2-8: induce back pBAD-BS; Swimming lane 9: standard molecular weight Marker.
Embodiment
Below in conjunction with embodiment the present invention is described in further details, but is not limited thereto.
All available from the precious biotechnology in Dalian company limited, 0.22um cellulose mixture film purifies device factory available from the new Asia, Shanghai City for expression plasmid pET-32a among the embodiment, escherichia coli DH5a, e. coli bl21 (DE3), TOP10 competent cell.
1) design of the plain peptide gene of capsule and synthetic and SOE-PCR amplification
According to the habitual codon of intestinal bacteria and two primers F 1 of BS gene order design, F2 by overlapping PCR method (SOE-PCR) amplification, obtains BS sample peptide gene fragment.This BS sample peptide gene fragment length is 99bp, is in series by the 10 BS gene orders (K-H-G) that copy, and the BS gene order upstream and downstream of 10 copies have EcoRI and SalI site site respectively.Through order-checking, this BS sample peptide gene fragment sequence is shown in SEQ ID NO.2.
Overlapping PCR reaction conditions: 94 ℃ of pre-sex change 3min enter PCR circulation: 94 ℃, and 45s, annealing temperature is from 58 ℃, every circulation 1.5min, totally 35 circulations; 68 ℃ are extended 10min, and the PCR product is identified through 1% agarose gel electrophoresis.The band (Fig. 1) of visible three treaty 100bp sizes behind the PCR product electrophoresis.
EcoRI
F1：5-CA
AAACACGGTAAGCACGGCAAACATGGTAAACACGGTAAGCATGGC-3(SEQ?ID?NO.3)
SalI
F2：5-G
TTAACCGTGTTTACCGTGCTTACCGTGTTTGCCATGTTTACCGTGCTTG-3(SEQ?ID?NO.4)
2) structure of the plain sample peptide of capsule recombinant expression vector
Respectively BS sample peptide gene fragment and expression vector pET-32a are carried out double digestion with nucleic acid restriction endonuclease EcoR I and SalI, the BS sample peptide gene fragment that the process enzyme is cut is spent the night for 4 ℃ by 1: 4 mol ratio with expression vector pET-32a and is connected, make up the plain sample peptide of capsule recombinant expression vector PET32a-BS, the BS sample peptidyl among this recombinant expression vector PET32a-BS is because the BS gene (Fig. 2) of 10 copies.
3) transformed into escherichia coli DH5a
Utilize CaCl
2Legal system is equipped with competent bacillus coli DH 5 alpha, gets above-mentioned recombinant expression vector PET32a-BS adding and contains in the polypropylene centrifuge tube of the competent bacillus coli DH 5 alpha of 50 μ L, gently ice bath 20min behind the mixing.Polypropylene centrifuge tube is taken out back 45 ℃ of heat-shocked 50sec from ice, then ice bath 1min immediately.The LB substratum that adds 30 ℃ of preheatings of 400 μ L is in 30 ℃ of jolting 1h.Get 150 μ L bacterium liquid and evenly be coated with the agar LB flat board that contains penbritin (Amp) 50 μ g/ml, behind 37 ℃ of placement 30min, be inverted and cultivate 20h.The picking positive strain extracts recombinant plasmid.CaCl
2Method adopts this area ordinary method operation.Above-mentioned steps also can be referring to the bacillus coli DH 5 alpha products instruction.
4) the plain sample inducing peptide of capsule is expressed
Recombinant plasmid transformed is entered in the e. coli bl21 (DE3), picking list colony inoculation is to filling in 5ml LB liquid nutrient medium (the 50 μ g/ml penbritin) test tube, spend the night in 30 ℃ of shaking table shaking culture, therefrom taking out thalline in second day is added to another and fills in the 10ml LB liquid nutrient medium, make cell concentration reach 0.1,30 ℃ of jolting of OD600 ≈ and cultivated 6～8 hours, when cell concentration OD600 ≈ 0.4～0.6, the adding final concentration is that the IPTG of 1mM carries out abduction delivering 6～8h, collects bacterium liquid.SDS-PAGE gel electrophoresis (Fig. 3) is carried out in sampling, and densitometric scan shows that BS content accounts for 28.6% of bacterial protein.
5) aftertreatment of the plain sample peptide of capsule
Through 4 ℃, the centrifugal 10min of 10000rpm/min collects bacterial sediment, with 0 ℃ of ice-cold high-permeation solution suspension bacterial sediment, mixes ice bath 10min with the plain sample peptide of the capsule bacterium liquid after expressing.Then, 4 ℃, the centrifugal 10min of 10000rpm/min collects bacterial sediment, with 0 ℃ of ice-cold hyposmosis solution of equal-volume bacterial sediment that suspends gently, ice bath placement 10min, and reversing centrifuge tube mixing frequently.4 ℃, the centrifugal 10min of 10000rpm/min; Then, the centrifuging and taking supernatant liquor is crossed 0.22um cellulose mixture film, obtains the plain tripeptides of capsule.
Described high-permeation solution: pH=8.5, final concentration Tris-HCl 25mmol/L, EDTA 2.5mmol/L, sucrose 25wt%; Described hyposmosis solution is: pH=8.5, final concentration Tris-HCl 25mmol/L, EDTA 2.5mmol/L.
Described LB nutrient media components is as follows:
Tryptone (Tryptones): 10g
Yeast Extract (yeast extract): 5g
NaCl (sodium-chlor): 10g
H
2O (water): 1000ml
The configuration solid medium then adds 15g Agar (agar) again.
Reference examples
Employing Li Ping etc. are at the plain tripeptides of the interim biological method synthetic capsular of mentioning of Chinese animal doctor's magazine 2006-42-4
1) designs and synthesizes the plain tripeptides gene fragment of capsule
According to colibacillary habitual codon 3 amino acid of capsule element are converted into nucleotide sequence and design following two gene fragment BS1 and BS2, article two, gene fragment is synthetic by Bo Ya bio-engineering corporation, in addition, synthetic one of downstream design according to the cloning site of pBAD/TOPO detects primer P1, whether match with inserting fragment BS1, it is correct to be used to detect direction of insertion.Fragment sequence is as follows.
BS1：5’-atgggtcataaaggtcataaaggtcataaaggtcataaaggtcataaaggtcataaaggtcataaa-3’(SEQ?ID?NO.5)
BS2：5’-tactttatgacctttatgacctttatgacctttatgattatgacctttatgacctttatgacc-3’(SEQ?ID?NO.6)
P1：5’-gcaaccaaagtgggtgcactg-3’(SEQ?ID?NO.7)
2) structure of expression vector
With PCR method amplification BS gene, primer is BS1 and BS2, with from as template.The PCR product is the cDNA fragment of 8 BS tripeptides of coding, and called after BS8, its expression product are BS8.The recovery of PCR product and purifying are undertaken by the glue purification test kit specification sheets of Omega company.Purifying reclaims the PCR product that obtains and is connected with the T-A tail with the pBAD/TOPO carrier.Connect product and transform the TOP10 competent cell, identify positive colony bacterium colony and called after pBAD-BS-TOP10 with PCR.
3) abduction delivering of gene fragment on pBAD/TOPO
Positive bacterial classification pBAD-BS-TOP10 is cultured to 0D in containing the LB substratum of Amp
600Value reaches at 0.5 o'clock, adds final concentration and is 0.02% the Arabic aldose of L-and carry out abduction delivering.SDS-PAGE gel electrophoresis (Fig. 4), densitometric scan show that the high expression level amount of BS accounts for 23.7% of bacterial protein.Experimental procedure in the reference examples can be operated by relevant open source literature.
Can find after testing, expressing gene of the present invention can efficiently express in host cell, BS content can account for 28.6% of bacterial protein, and expression amount is higher than in the prior art 23.7% of high expression level amount far away, and making the plain tripeptides of capsule carry out suitability for industrialized production with biological synthesis process becomes possibility.
BS sample peptide gene fragment in the present embodiment is according to sequence synthetic shown in the SEQ ID NO.2 by Shandong Sinostar Bioscience and Technology Co., Ltd..
As described in embodiment 1, difference is that the Connection Step of BS sample peptide gene fragment and expression vector pET-32a is as follows:
Respectively BS sample peptide gene fragment and expression vector pET-32a are carried out double digestion with EcoR I and Xho I, it is composed as follows that enzyme is cut system:
ddH
2O 34.0μL ddH
2O 34.0μL
10×H?Buffer 5.0μL 10×H?Buffer 5.0μL
BS sample peptide gene fragment 10.0 μ L expression vector pET-32a 10.0 μ L
EcoR?I 0.5μL EcoR?I 0.5μL
Xho?I 0.5μL Xho?I 0.5μL
The above-mentioned reaction system of mixing, and place 37 ℃ of water-bath effect 3h, enzyme to cut product equally after 1.5% agarose gel electrophoresis is identified, glue reclaims test kit and reclaims evaluation.
The expression vector pET-32a of the BS sample peptide gene fragment of process double digestion and same process double digestion spends the night for 4 ℃ by 1: 2.5 mol ratio and is connected, and makes up the plain sample peptide of capsule recombinant expression vector PET32a-BS.
After testing, the high expression level amount of BS accounts for 30.6% of bacterial protein.
SEQUENCE?LISTING
＜110〉Shandong Sinostar Bioscience and Technology Co., Ltd.
＜120〉the plain sample peptide of a kind of genetically engineered capsule and expressing gene and application
<160>7
<170>PatentIn?version?3.5
<210>1
<211>30
<212>PRT
＜213〉synthetic
<400>1
Lys?His?Gly?Lys?His?Gly?Lys?His?G1y?Lys?His?Gly?Lys?His?Gly?Lys
1 5 10 15
His?Gly?Lys?His?Gly?Lys?His?Gly?Lys?His?Gly?Lys?His?Gly
20 25 30
<210>2
<211>99
<212>DNA
＜213〉synthetic
<400>2
aagcatggaa?agcatggaaa?gcatggaaag?catggaaagc?atggaaagca?tggaaagcat 60
ggaaagcatg?gaaagcatgg?aaagcatgga?aagcatgga 99
<210>3
<211>53
<212>DNA
＜213〉synthetic
<400>3
cagaattcaa?acacggtaag?cacggcaaac?atggtaaaca?cggtaagcat?ggc 53
<210>4
<211>56
<212>DNA
＜213〉synthetic
<400>4
ggtcgactta?accgtgttta?ccgtgcttac?cgtgtttgcc?atgtttaccg?tgcttg 56
<210>5
<211>66
<212>DNA
＜213〉synthetic
<400>5
atgggtcata?aaggtcataa?aggtcataaa?ggtcataaag?gtcataaagg?tcataaaggt 60
cataaa 66
<210>6
<211>63
<212>DNA
＜213〉synthetic
<400>6
tactttatga?cctttatgac?ctttatgacc?tttatgatta?tgacctttat?gacctttatg 60
acc 63
<210>7
<211>21
<212>DNA
＜213〉synthetic
<400>7
gcaaccaaag?tgggtgcact?g 21
Claims (10)
1. the plain sample peptide of genetically engineered capsule has aminoacid sequence shown in SEQ ID NO.1.
2. the gene fragment of the plain sample peptide of the described genetically engineered capsule of the claim 1 of encoding has nucleotide sequence shown in SEQ ID NO.2.
3. expression vector that contains nucleotide sequence shown in the SEQ ID NO.2.
4. the application of the gene fragment of the plain sample peptide of the described genetically engineered capsule of claim 2 in the plain tripeptides of preparation capsule.
5. application as claimed in claim 4 is characterized in that step is as follows:
(1) gene fragment of the plain sample peptide of genetically engineered capsule is inserted expression plasmid, obtain recombinant plasmid;
(2) the recombinant plasmid transformed host bacterium that step (1) is made makes up host bacterium expression system;
(3) the plain sample peptide of the host bacterium expression system abduction delivering capsule that utilizes step (2) to make up, and process aftertreatment technology makes the plain tripeptides of capsule.
6. application as claimed in claim 5, it is characterized in that, described in the step (1) gene fragment of the plain sample peptide of genetically engineered capsule is inserted expression plasmid, be meant gene fragment with nucleic acid restriction endonuclease EcoR I and the plain sample peptide of one of SalI, Xho I or HindIII double digestion genetically engineered capsule, be connected on the expression plasmid that same enzyme cuts, carry out sequence verification then.
7. application as claimed in claim 6 is characterized in that, described expression plasmid is: pET-32a, pET-32b or pET-32c.
8. application as claimed in claim 5 is characterized in that, the host bacterium is described in the step (2): e. coli bl21 (DE3).
9. application as claimed in claim 5 is characterized in that, the step of aftertreatment technology is as follows in the step (3):
The plain sample peptide of capsule after expressing bacterium liquid after centrifugal, is collected bacterial sediment,, mix ice bath 8～10min with 0 ℃ of ice-cold high-permeation solution suspension bacterial sediment; Then, centrifugal collection bacterial sediment with 0 ℃ of ice-cold hyposmosis solution suspension bacterial sediment of equal-volume, mixes ice bath 8～10min; Then, the centrifuging and taking supernatant liquor is crossed 0.2～0.24um cellulose mixture film, obtains the plain tripeptides of capsule.
10. application as claimed in claim 9 is characterized in that, described high-permeation solution: pH=8.5, final concentration Tris-HCl20～30mmol/L, EDTA 2～3mmol/L, sucrose 25wt%; Described hyposmosis solution is: pH=8.5, final concentration Tris-HCl 20～30mmol/L, EDTA 2～3mmol/L.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2010101902669A CN101880314B (en) | 2010-06-03 | 2010-06-03 | Bursin-like peptide of gene engineering as well as expression gene and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN2010101902669A CN101880314B (en) | 2010-06-03 | 2010-06-03 | Bursin-like peptide of gene engineering as well as expression gene and application thereof |
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| Publication Number | Publication Date |
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| CN101880314A true CN101880314A (en) | 2010-11-10 |
| CN101880314B CN101880314B (en) | 2012-11-28 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102178950A (en) * | 2011-05-06 | 2011-09-14 | 河南科技大学 | Subunit vaccine immunologic adjuvant and application thereof |
| CN102225195A (en) * | 2011-06-15 | 2011-10-26 | 山东华辰生物科技有限公司 | Porcine interferon-gamma composite medicament and application thereof |
| CN102698264A (en) * | 2012-02-07 | 2012-10-03 | 江苏省农业科学院 | Bursin-like polypeptide derivative applied as immuno-enhancer of compound hog cholera vaccines |
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| CN1696151A (en) * | 2005-05-19 | 2005-11-16 | 四川键宇胚胎工程有限公司 | Method for synthesizing bursa of Fabricius bursin |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1696151A (en) * | 2005-05-19 | 2005-11-16 | 四川键宇胚胎工程有限公司 | Method for synthesizing bursa of Fabricius bursin |
Non-Patent Citations (3)
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| 《中国兽医杂志》 20061231 李萍等 囊素三肽的基因克隆和表达 第7-10页 1-10 第42卷, 第4期 2 * |
| 《江苏农业学报》 20031231 张德明等 法氏囊提取物中囊素三肽含量的测定 第123-124页 1-10 第19卷, 第2期 2 * |
| 《郑州牧业工程高等专科学校学报》 20010531 刘兴友 囊素三肽研究进展 第101-104页 1-10 第21卷, 第2期 2 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102178950A (en) * | 2011-05-06 | 2011-09-14 | 河南科技大学 | Subunit vaccine immunologic adjuvant and application thereof |
| CN102225195A (en) * | 2011-06-15 | 2011-10-26 | 山东华辰生物科技有限公司 | Porcine interferon-gamma composite medicament and application thereof |
| CN102225195B (en) * | 2011-06-15 | 2013-01-02 | 山东华辰生物科技有限公司 | Porcine interferon-gamma composite medicament and application thereof |
| CN102698264A (en) * | 2012-02-07 | 2012-10-03 | 江苏省农业科学院 | Bursin-like polypeptide derivative applied as immuno-enhancer of compound hog cholera vaccines |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101880314B (en) | 2012-11-28 |
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