CN113045671A - Recombinant antigen for detecting schistosoma japonicum, preparation method and application thereof - Google Patents

Recombinant antigen for detecting schistosoma japonicum, preparation method and application thereof Download PDF

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CN113045671A
CN113045671A CN202110127995.8A CN202110127995A CN113045671A CN 113045671 A CN113045671 A CN 113045671A CN 202110127995 A CN202110127995 A CN 202110127995A CN 113045671 A CN113045671 A CN 113045671A
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王竑婷
王立坚
王晓明
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Changchun Wan Cheng Bio Electron Co ltd
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Changchun Wan Cheng Bio Electron Co ltd
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Abstract

The invention relates to the field of biotechnology, in particular to a recombinant antigen for detecting schistosoma japonicum. The recombinant antigen comprises a GST protein fragment, a rSjGCP protein fragment and a Sj23 protein fragment of schistosoma japonicum which are connected in sequence from an N end to a C end. Compared with the existing GST antigen, the recombinant antigen disclosed by the invention has the advantages that the immunogenicity of the antigen is improved to the maximum extent, the detection sensitivity is improved, the water solubility of the recombinant antigen is higher, and the purification efficiency is higher.

Description

Recombinant antigen for detecting schistosoma japonicum, preparation method and application thereof
Technical Field
The invention relates to the field of biotechnology, in particular to a recombinant antigen for detecting schistosoma japonicum, a preparation method and application thereof.
Background
Schistosoma japonicum, also known as schistosoma japonicum, is parasitic on the human body, there are three main types of schistosoma japonicum: schistosoma japonicum, which is prevalent in the northern part of africa; schistosoma mansoni, prevalent in Latin America and in the middle of Africa, and Schistosoma japonicum, prevalent in Asia, cause mechanical damage to the host.
At present, the methods for diagnosing schistosomiasis mainly comprise etiological diagnosis, immunological diagnosis, ultrasonic diagnosis and the like. The traditional etiology diagnosis method is the most reliable and classical method for diagnosing schistosomiasis, is the gold standard for judging whether schistosomiasis is ill or not, and particularly relates to a Kato-Katz smear manure method and a miracidium incubation method MHT, but the two methods have the problems of time and labor waste and poor compliance of the public. Immunological diagnosis has become a common diagnostic method for schistosomiasis at present due to its advantages of good sensitivity, specificity and practicability, and mainly comprises: indirect Hemagglutination Assay (IHA), immunoenzyme-linked immunosorbent assay (ELISA), colloidal gold labeling technology, and the like. However, the existing Indirect Hemagglutination Assay (IHA) (ELISA) still generally has the problems of long detection time and insufficient sensitivity, and the antigen adopted by the detection method taking the immune enzyme-linked adsorption assay and the immune colloidal gold technology as the core is generally GST, which has poor immunogenicity and low sensitivity.
Disclosure of Invention
In view of the above, the present invention provides a recombinant antigen for detecting schistosoma japonicum, a preparation method and applications thereof. The recombinant antigen fuses partial fragments of GST, rSjGCP and Sj23, has stronger immunogenicity, and has higher sensitivity for detecting the schistosoma japonicum compared with the existing antigen.
The invention provides a recombinant antigen for detecting schistosoma japonicum, which comprises a GST protein fragment, a rSjGCP protein fragment and a Sj23 protein fragment of schistosoma japonicum which are connected in sequence from an N end to a C end.
In some embodiments, the amino acid sequence of the GST protein fragment is shown in SEQ ID NO.1, the amino acid sequence of the rSjGCP protein fragment is shown in SEQ ID NO.2, and the amino acid sequence of the Sj23 protein fragment is shown in SEQ ID NO. 3.
In some embodiments, the GST protein fragment and the rSjGCP protein fragment are linked by a linker peptide 1 having an amino acid sequence shown in SEQ ID No.4, and the rSjGCP protein fragment and the Sj23 protein fragment are linked by a linker peptide 2 having an amino acid sequence shown in SEQ ID No. 5.
In some embodiments, the amino acid sequence of the recombinant antigen is set forth in SEQ ID No.6, and the nucleotide sequence encoding the recombinant antigen is set forth in SEQ ID No. 7.
In some embodiments, the C-terminus of the Sj23 protein fragment is further linked to an Sj28 protein fragment. In some embodiments, the amino acid sequence of the Sj28 protein fragment is shown in SEQ ID NO. 8. The Sj23 protein fragment and the Sj28 protein fragment are connected through a connecting peptide 2 with an amino acid sequence shown as SEQ ID NO. 5.
In some embodiments, the amino acid sequence of the recombinant antigen is shown as SEQ ID NO.9, and the nucleotide sequence encoding the recombinant antigen is shown as SEQ ID NO. 10.
The present invention queries and downloads GST, Sj23, rSjGcp and Sj28 protein sequences through NCBI (national Center for Biotechnology information) national Center for Biotechnology information database. Wherein, GST protein sequence number: 1Y6E _ B, Sj23 protein sequence number: AAA29920, rSjGcp protein sequence number: AAN39279, Sj28 protein sequence AAA29892, partial sequences (hydrophilic region and region with higher immunogenicity) in four protein sequences are intercepted, synthesized and spliced to obtain recombinant antigen, and the amino acid sequences are shown in SEQ ID NO.6 and SEQ ID NO. 9.
The recombinant antigen is expressed by an escherichia coli protein expression system as a basis, and is purified by GSH resin gel to obtain the recombinant antigen which can be used for rapidly detecting the anti-schistosome antibody in an infected body. Through detection, the detection sensitivity of the recombinant antigen detection of the invention on the antibody in an infected body is 100%, and the detection specificity of an uninfected sample and infected fasciola gigantica is 93.7%. The detection result of the existing GST antigen is that the sensitivity is 79 percent, the specificity is 81 percent, and both the sensitivity and the specificity are obviously superior to the existing antigen.
The invention also provides the application of the recombinant antigen in preparing a product for detecting the schistosoma japonicum.
The invention also provides a DNA molecule encoding the recombinant antigen. In some embodiments, the nucleotide sequence of the DNA molecule encoding the recombinant antigen is shown in SEQ ID NO.9 or SEQ ID NO. 10.
The invention also provides a recombinant vector containing the DNA molecule.
In some embodiments, the backbone vector of the recombinant vector is pGEX-4T-2.
The invention also provides a host cell transformed with the recombinant vector.
In some embodiments, the host cell is e. In some embodiments, the host cell is e.coli BL21 (D3).
The invention provides a recombinant antigen for detecting schistosoma japonicum, which comprises a GST protein fragment, a rSjGCP protein fragment and a Sj23 protein fragment of schistosoma japonicum which are connected in sequence from an N end to a C end. Compared with the existing GST antigen, the recombinant antigen of the invention improves the immunogenicity of the antigen to the maximum extent and improves the sensitivity and specificity of detection, wherein the sensitivity of antibody detection in infected serum is 100 percent, and the detection specificity of uninfected samples and infected fasciola gigantica is 93.7 percent.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows a map of a recombinant vector containing a DNA molecule encoding recombinant antigen 1;
FIG. 2 shows a map of a recombinant vector containing a DNA molecule encoding recombinant antigen 2.
Detailed Description
The invention discloses a recombinant antigen for detecting schistosoma japonicum, a preparation method and application thereof, and a person skilled in the art can realize the detection by properly improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The recombinant antigen for detecting schistosoma japonicum, the preparation method and the raw materials and reagents used in the application of the recombinant antigen provided by the invention can be purchased from the market.
The invention is further illustrated by the following examples:
example 1
The recombinant antigen 1 comprises a GST protein fragment (SEQ ID NO.1), a rSjGCP protein fragment (SEQ ID NO.2) and a Sj23(SEQ ID NO.3) protein fragment of schistosoma japonicum which are sequentially connected from an N end to a C end, the GST protein fragment and the rSjGCP protein fragment are connected through a connecting peptide 1(SEQ ID NO. 4: SDLEVLFQGPRGS) with an amino acid sequence shown as SEQ ID NO.4, the rSjGCP protein fragment and the Sj23 protein fragment are connected through a connecting peptide 2(SEQ ID NO. 5: GGGGSGGGGS) with an amino acid sequence shown as SEQ ID NO.5, and the amino acid sequence of the rSjGCP protein fragment is shown as SEQ ID NO: 6:
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPRGSMRIGYEGLPRDQWPKVIHWNLHARDGIIWVLDGLLKCPEKLCPLLAEDVDYYGGGGSGGGGSMTGALENPNEEITATMDKIQTSFHCCGVKGPDDYKGNVPASCKEGQEVYVQGCLSVFSAFLKRN
the nucleotide sequence for coding the recombinant antigen 1 is shown as SEQ ID NO: shown at 7.
The recombinant antigen 2 comprises a GST protein fragment, a rSjGCP protein fragment, a Sj23 protein fragment and a Sj28 protein fragment (SEQ ID NO.8) of schistosoma japonicum which are connected in sequence from an N end to a C end, and the amino acid sequence of the recombinant antigen is shown in SEQ ID NO: 9:
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPRGSMRIGYEGLPRDQWPKVIHWNLHARDGIIWVLDGLLKCPEKLCPLLAEDVDYYGGGGSGGGGSMTGALENPNEEITATMDKIQTSFHCCGVKGPDDYKGNVPASCKEGQEVYVQGCLSVFSAFLKRNGGGGSGGGGSMKPEEEKQKIIKEILNGKVPVLLDIICESLKASTGKLAV
the nucleotide sequence encoding the recombinant antigen 2 is shown in SEQ ID NO: 10.
respectively adopting gene cloning technology to make SEQ ID NO: 7. SEQ ID NO: the gene fragment of 10 is transferred into pGEX-4T-2 plasmid vector (recombinant vector is shown in figure 1 and figure 2), then is transferred into BL21(D3) strain, uses IPTG as inducer to promote the bacteria to express a large amount of target protein, and is purified by GSH resin. The specific method comprises the following steps:
LB medium, 100ug/ml ampicillin, was added to the Erlenmeyer flask. 1: the recombinant gene-transformed bacterium BL21 was cultured overnight at 500 volumes, shake-cultured overnight at 37 ℃ and then induced by the addition of isopropyl β -D-thiogalactoside (IPTG), and further cultured at 37 ℃ for 1 hour and centrifuged to collect the bacterium. Adding lysis solution to lyse bacteria, centrifuging to collect supernatant, adding the supernatant into a purification medium containing GST tag protein, washing the column with PBS and GST eluent buffer solution in sequence, and finally collecting elution buffer solution which contains target protein. Bacteria with the dry weight of 1mg can generate 100ug of recombinant protein, wherein most of the protein can not be purified by forming inclusion body precipitates, only 15-25% of the protein can be purified by dissolving in a lysate, about 10-20 ug of target protein can be obtained by purification, the total purification efficiency of the recombinant antigen 1 (the amino acid sequence is shown as SEQ ID NO. 7) is 20%, and the purification efficiency of the relative soluble protein is 80%. The total purification efficiency of the recombinant antigen 2 (the amino acid sequence is shown as SEQ ID NO.8) is 10 percent, and the relative purification efficiency of the soluble protein is 67 percent.
Example 2
50 rabbits were taken, infected with 300 Japanese blood fluke, and then treated with praziquantel (80 mg/kg body weight) 6 weeks after infection. Blood samples were collected pre-infection, 2 weeks, 4 weeks, 6 weeks post-infection, and 2 weeks, 4 weeks, and 6 weeks post-praziquantel treatment. In addition, 20 rabbit sera infected with fasciola gigantica were collected as a reference in the same manner, and 20 rabbit sera not infected were collected as a negative control. 100ul of PBS (pH 7.6) containing 20ug/ml of recombinant antigen 1 represented by SEQ ID NO.7, recombinant antigen 2 represented by SEQ ID NO.8 or GST protein was added to a polystyrene 96 microplate to coat the wells overnight at 4 ℃, and then the concentration of the protein was measured as 1: 100 dilutions of rabbit serum were incubated for 1 hour at 37 ℃. Goat anti-mouse IgG conjugated horseradish peroxidase antibody was added and incubated at 37 ℃ for 3 hours, followed by washing 3 times with 200ul of 10% Tween-containing PBS per well. After the reaction of the reaction substrate o-phenylenediamine with hydrogen peroxide for 10 minutes, 2N H2SO4 was added to terminate the reaction. The plate was read at 490nm using an enzyme linked immunosorbent assay and the results were determined.
The results show that: the sensitivity of the antibody detection of the recombinant antigen 2 in the infected serum is 100 percent respectively, and the detection specificity of the uninfected sample and the infected fasciola gigantica is 93.7 percent respectively. The GST detection result showed 79% sensitivity and 81% specificity.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60
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<210> 8
<211> 126
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Ala Thr Gly Ala Ala Ala Cys Cys Gly Gly Ala Ala Gly Ala Ala Gly
1 5 10 15
Ala Ala Ala Ala Ala Cys Ala Gly Ala Ala Ala Ala Thr Cys Ala Thr
20 25 30
Cys Ala Ala Ala Gly Ala Gly Ala Thr Cys Cys Thr Gly Ala Ala Cys
35 40 45
Gly Gly Cys Ala Ala Ala Gly Thr Thr Cys Cys Gly Gly Thr Thr Cys
50 55 60
Thr Gly Cys Thr Gly Gly Ala Thr Ala Thr Cys Ala Thr Cys Thr Gly
65 70 75 80
Cys Gly Ala Ala Ala Gly Cys Cys Thr Gly Ala Ala Ala Gly Cys Ala
85 90 95
Ala Gly Cys Ala Cys Cys Gly Gly Thr Ala Ala Ala Cys Thr Gly Gly
100 105 110
Cys Ala Gly Thr Gly Thr Ala Ala Gly Ala Ala Thr Thr Cys
115 120 125
<210> 9
<211> 406
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro
1 5 10 15
Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu
20 25 30
Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu
35 40 45
Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys
50 55 60
Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn
65 70 75 80
Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu
85 90 95
Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser
100 105 110
Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu
115 120 125
Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn
130 135 140
Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp
145 150 155 160
Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu
165 170 175
Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr
180 185 190
Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala
195 200 205
Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Glu Val Leu
210 215 220
Phe Gln Gly Pro Arg Gly Ser Met Arg Ile Gly Tyr Glu Gly Leu Pro
225 230 235 240
Arg Asp Gln Trp Pro Lys Val Ile His Trp Asn Leu His Ala Arg Asp
245 250 255
Gly Ile Ile Trp Val Leu Asp Gly Leu Leu Lys Cys Pro Glu Lys Leu
260 265 270
Cys Pro Leu Leu Ala Glu Asp Val Asp Tyr Tyr Gly Gly Gly Gly Ser
275 280 285
Gly Gly Gly Gly Ser Met Thr Gly Ala Leu Glu Asn Pro Asn Glu Glu
290 295 300
Ile Thr Ala Thr Met Asp Lys Ile Gln Thr Ser Phe His Cys Cys Gly
305 310 315 320
Val Lys Gly Pro Asp Asp Tyr Lys Gly Asn Val Pro Ala Ser Cys Lys
325 330 335
Glu Gly Gln Glu Val Tyr Val Gln Gly Cys Leu Ser Val Phe Ser Ala
340 345 350
Phe Leu Lys Arg Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met
355 360 365
Lys Pro Glu Glu Glu Lys Gln Lys Ile Ile Lys Glu Ile Leu Asn Gly
370 375 380
Lys Val Pro Val Leu Leu Asp Ile Ile Cys Glu Ser Leu Lys Ala Ser
385 390 395 400
Thr Gly Lys Leu Ala Val
405
<210> 10
<211> 1212
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60
ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120
tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180
ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240
atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300
gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360
gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420
acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480
gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540
aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600
tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660
ctggttccgc gtggatccat gcgtattggt tacgaaggtc tgccgcgcga tcagtggccg 720
aaagtcattc attggaacct gcacgcacgc gacggtatta tttgggttct ggacggcctg 780
ctgaaatgtc cggaaaaact gtgtccgctg ctggcagaag acgttgatta ttatggcggc 840
ggcggttctg gcggcggtgg tagtatgacc ggcgcactgg aaaatccgaa cgaagagatc 900
accgcgacca tggacaaaat ccagaccagc tttcattgtt gcggcgttaa aggcccggac 960
gattacaaag gcaacgttcc ggcaagctgc aaagaaggtc aagaagttta cgtgcagggt 1020
tgtctgagcg tttttagcgc gtttctgaaa cgtaacggcg gcggcggttc tggtggcggc 1080
ggtagtatga aaccggaaga agaaaaacag aaaatcatca aagagatcct gaacggcaaa 1140
gttccggttc tgctggatat catctgcgaa agcctgaaag caagcaccgg taaactggca 1200
gtgtaagaat tc 1212

Claims (15)

1. A recombinant antigen for detecting schistosoma japonicum is characterized by comprising a GST protein fragment, a rSjGCP protein fragment and a Sj23 protein fragment of schistosoma japonicum which are connected in sequence from an N end to a C end.
2. The recombinant antigen according to claim 1, wherein the amino acid sequence of the GST protein fragment is shown as SEQ ID NO.1, the amino acid sequence of the rSjGCP protein fragment is shown as SEQ ID NO.2, and the amino acid sequence of the Sj23 protein fragment is shown as SEQ ID NO. 3.
3. The recombinant antigen according to claim 1, wherein the GST protein fragment and the rSjGCP protein fragment are connected by a linker peptide 1 having an amino acid sequence shown in SEQ ID No.4, and the rSjGCP protein fragment and the Sj23 protein fragment are connected by a linker peptide 2 having an amino acid sequence shown in SEQ ID No. 5.
4. The recombinant antigen according to claim 3, wherein the amino acid sequence of the recombinant antigen is as shown in SEQ ID No. 6.
5. The recombinant antigen of any one of claims 1 to 4, wherein the C-terminus of the Sj23 protein fragment is further connected with an Sj28 protein fragment.
6. The recombinant antigen according to claim 5, wherein the amino acid sequence of the Sj28 protein fragment is shown in SEQ ID NO. 8.
7. The recombinant antigen of claim 5, wherein the Sj23 protein fragment and the Sj28 protein fragment are connected by a connecting peptide 2 with an amino acid sequence shown as SEQ ID NO. 5.
8. The recombinant antigen according to claim 5, wherein the amino acid sequence of the recombinant antigen is shown as SEQ ID No. 9.
9. Use of the recombinant antigen of any one of claims 1 to 8 in the preparation of a product for the detection of schistosoma japonicum.
10. A DNA molecule encoding the recombinant antigen of any one of claims 1-8.
11. The DNA molecule according to claim 10, characterized in that its nucleotide sequence is as shown in SEQ ID No.7 or SEQ ID No. 10.
12. A recombinant vector comprising the DNA molecule of claim 10 or 11.
13. The recombinant vector according to claim 12, wherein the backbone vector of the recombinant vector is pGEX-4T-2.
14. A host cell transformed with the recombinant vector of claim 12 or 13.
15. The host cell of claim 14, wherein the host cell is e.
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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1162017A (en) * 1996-04-08 1997-10-15 中国农业科学院上海家畜寄生虫病研究所 Systematic production of recombined Nippon blood-flukes glutathione transferase by using domestic silk-worm
EP0992582A2 (en) * 1998-10-07 2000-04-12 University of Wales, Bangor Schistosoma recombinant fusion protein vaccine
CN1422953A (en) * 2002-01-28 2003-06-11 中国农业科学院上海家畜寄生虫病研究所 Gene sequence of Japanese shistosome China mainland strain gynecophoric canal protein
US20040029781A1 (en) * 2002-06-12 2004-02-12 Sigma-Aldrich Co. Affinity peptides and method for purification of recombinant proteins
CN101444622A (en) * 2008-06-06 2009-06-03 中国人民解放军军事医学科学院军事兽医研究所 Schistosoma japonicum gene recombination living vaccine production technique taking porcine pseudorabies virus as vector and product
CN101624422A (en) * 2008-07-09 2010-01-13 中国农业科学院上海兽医研究所 Schistosoma japonicum recombinant multi-epitope antigens, method for expressing and purifying same and application thereof
CN102040658A (en) * 2009-10-19 2011-05-04 中国农业科学院上海兽医研究所 Schistosoma japonica polypeptide with immunogenicity and application thereof
CN103059117A (en) * 2011-10-21 2013-04-24 同济大学 High throughput screening of important schistosoma japonicum antigens and application of antigens in schistosomiasis diagnosis
CN103405779A (en) * 2013-07-18 2013-11-27 江苏省血吸虫病防治研究所 Long-acting artesunate drug for preventing schistosome infection and preparation method thereof

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1162017A (en) * 1996-04-08 1997-10-15 中国农业科学院上海家畜寄生虫病研究所 Systematic production of recombined Nippon blood-flukes glutathione transferase by using domestic silk-worm
EP0992582A2 (en) * 1998-10-07 2000-04-12 University of Wales, Bangor Schistosoma recombinant fusion protein vaccine
CN1422953A (en) * 2002-01-28 2003-06-11 中国农业科学院上海家畜寄生虫病研究所 Gene sequence of Japanese shistosome China mainland strain gynecophoric canal protein
US20040029781A1 (en) * 2002-06-12 2004-02-12 Sigma-Aldrich Co. Affinity peptides and method for purification of recombinant proteins
CN101444622A (en) * 2008-06-06 2009-06-03 中国人民解放军军事医学科学院军事兽医研究所 Schistosoma japonicum gene recombination living vaccine production technique taking porcine pseudorabies virus as vector and product
CN101624422A (en) * 2008-07-09 2010-01-13 中国农业科学院上海兽医研究所 Schistosoma japonicum recombinant multi-epitope antigens, method for expressing and purifying same and application thereof
CN102040658A (en) * 2009-10-19 2011-05-04 中国农业科学院上海兽医研究所 Schistosoma japonica polypeptide with immunogenicity and application thereof
CN103059117A (en) * 2011-10-21 2013-04-24 同济大学 High throughput screening of important schistosoma japonicum antigens and application of antigens in schistosomiasis diagnosis
CN103405779A (en) * 2013-07-18 2013-11-27 江苏省血吸虫病防治研究所 Long-acting artesunate drug for preventing schistosome infection and preparation method thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHAOLV等: "A perspective for improving the sensitivity of detection: The application of multi-epitope recombinant antigen in serological analysis of buffalo schistosomiasis", 《ACTA TROPICA》 *
CHENGCHEN等: "Reviews and advances in diagnostic research on Schistosoma japonicum", 《ACTA TROPICA》 *
张兆松等: "日本血吸虫重组抗原体外诱导细胞免疫应答的研究", 《中国动物学会第八次全国寄生虫学学术讨论会论文摘要汇编》 *

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