Conformation-specific monoclonal antibodies recognizing the native structure of G protein-coupled receptor (GPCR)
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Sf9
Immunofluorescence
Hybridoma technology
Using a hybrid baculovirus system, we compared the expression of 45 recombinant proteins from six categories using two models: silkworm (larvae and pupae) and an Sf9 cell line. A total of 45 proteins were successfully expressed; preparation of hybrid baculovirus was unsuccessful for one protein, and two proteins were not expressed. A similar pattern of expression was seen in both silkworm and Sf9 cells, with double and multiple bands found in immunoblotting of the precipitate of both hosts. Degraded proteins were seen only in the silkworm system (particularly in the larvae). Production was more efficient in silkworms; a single silkworm produced about 70 times more protein than 10(6) Sf9 cells in 2 ml of culture medium.
Sf9
Baculoviridae
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Objective To construct the recombinant EBV LMP1 baculovirus expression plasmid and express LMP1 in Sf9 cell line.Methods The EBV LMP1 cDNA was cloned into a pF AST B AC HTb donor plasmid, and then the recombinant plasmid was transformed into DH10BAC competent cells. The transformant containing the recombinant bacmid was selected and named as Bacmid/LMP1. The recombinant baculoviruses were obtained after the transfection of Bacmid/LMP1 into Sf9 lines. The techniques of IFA, SDS PAGE and Western blot were used to detect and identify the products expressed in Sf9 lines.Results The recombinant baculavirus was obtained. The recombinant LMP1 protein was expressed in Sf9 insect cells and detected by IFA and Western blot using monoclonal antibody CS1 4.The MW of the recombinant LMP1 protein was 63kDa, and could be detected in the supernatant of Sf9 cell culture.Conclusions Recombinant LMP1 could be expressed in Sf9 lines using the baculovirus expression system, and thus provided the basic material for studying of the function of LMP1 in the cacinogenesis of EBV and its application in immunotherapy.
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IGF-1 gene was amplified using PCR and cloned into pFastHTA with the EcoRⅠ and SalⅠ sites. The constructed pFast/IGF-1 was transformed into Escherichia coli DH10Bac. After screening with kanamycin, tetracycline, gentamicin and X-gal/IPTG, the recombinant baculovirus expressive vector pBacmid-Fa-IGF-1 was obtained. Then the sf9 cells were transfected using the recombinant pBacmid-Fa-IGF-1. After packing in the sf9 cells, recombinant viruses were obtained, which could be observed under electron microscope. A band of protein of about 13.0 kD was identified using Western-blot, which demonstrated that the IGF-1 was successfully expressed in the sf9 cells. The cell growth promotion rate was 24.4% measured using MTT method.
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Kanamycin
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Monoclonal antibodies are essential tools in molecular and cellular immunology research. They have essentially replaced the polyclonal antibodies in identifying blood groups and detecting cell markers and pathogenic agents. The aim of the present study is to produce monoclonal antibody identifying the ABO blood groups using the murine hybridoma technology. An anti-A monoclonal antibody A907 was selected and estimated for its use in the manufacture of a reagent anti-A. The selected antibody specifically reacts with A1, A2, A1B and A2B erythrocytes. It does not recognize B, O, A3 and Ax erythrocytes. The A907 monoclonal antibody can be used in blood grouping in association with a reagent recognizing the A weak phenotypes.
Polyclonal antibodies
Hybridoma technology
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Objective To obtain human Legumain protein with insect-baculovirus expression system,make foundation for the uveal melanoma study.Methods The gene encoding Legumain protein was cloned and inserted into the pFastBac vector,and the recombinant bacmid was generated after the E.coli DH10Bac competent cell was transformed by the recombinant pFastBac vector.The recombinant baculovirns was transfected into insect cell Sf9.Western Blotting and SDS-PAGE analysis of recombinant protein expression was performed.Results Recombinant baculovirns expressing Legumain protein was obtained.Western Blotting and SDS-PAGE analysis showed that Legumain protein was expressed when the Sf9 cells were infected by the recombinant baculovirus,which consistent with the expected results.Conclusions Legumain protein was obtained through insect-baculovirus expression system.Cloning and expression of Legumain protein provide the foundation for the mechanism of Legumain in the uveal melanoma invasion and metastasis.
Key words:
Baculovirus; Expression system; Insect cell Sf9; Legumain protein
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Cloning (programming)
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This research was focused on expressing reovirus type-3(Reo-3) S1 gene in Sf9 insect cells by Bac-to-Bac Baculovirus expression system.S1 gene was amplified by polymerase chain reaction(PCR) and inserted into pFastBacHTA to obtain recombinant transfer vector pFastBacHTA-S1.Recombinant Bacmid Bacmid-S1 was obtained after transformed pFastBacHTA-S1 into DH10Bac cells.Then,the Bacmid-S1 was transfected into insect cells through lipid-mediated transfection.Immunoflorescence and western blot analysis confirmed that the recombinant protein was expressed in Sf9 cells(50 kD) and had its immunogenicity.Based on our knowledge,this is the first report in China which shows Reo-3 σ1 protein with immunogenicity expressed in Sf9 insect cells successfully.
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[Objective] This study is to express Norovirus(GenegroupⅡ) VP2 in insect cells,analysis the subcellular localization of VP2 and lay the foundation of the function study of VP2.[Methods] Norovirus(GenegroupⅡ) ORF3 gene was amplified from plasmid pMD-ORF3 using primers P1/P2(including 6×His tag coding sequence).The product was cloned into the vector pFastBac1 to construct recombinant plasmid pFB-ORF3.pFB-ORF3 was transformed into competent DH10Bac to get recombinant bacmid Bac-ORF3.Recombinant baculovirus,Ac-VP2,was generated for expressing VP2,by transfecting recombinant Bac-ORF3 with LipofactamineTM 2000 into sf9 insect cells.The expression product was testified by the western blot and indirect immunofluorescence assay with monoclonal antibody against 6×His tag.[Results] The result of western blot showed a 29 kD specific bind in sf9 cells infected by Ac-VP2.Indirect im-munofluorescence assay showed the specific green fluorescence in sf9 cells infected by Ac-VP2,and the green fluorescence could localize at nuclear and membrane of sf9 cells.[Conclusion] These results demonstrated that the Norovirus(Gengroup Ⅱ) VP2 was expressed in sf9 cells successfully and could localize at nuclear and membrane of sf9 cells.
Sf9
Immunofluorescence
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Objective To construct human KCTD9 protein in sf9 expression system and harvest the protein for further functional study. Methods To express hKCTD9 in insect cells,the full length hKCTD9 gene was obtained from pMD18-T-hKCTD9 plasmid. The gene was then inserted into pENTR/D-TOPO plasmid. After homologous recombination of the plasmid with BaculoDirectTM linear DNA,the recombinant baculovirus DNA containing the hKCTD9 gene fragment was obtained. Sf9 cells were transfected with baculovirus DNA via Cellfectin and cultured for 3 days. After 3 rounds amplification of recombinant baculovirus,the expression of hKCTD9 in sf9 cells was detected and identified by the laser confocal microscopy and Western blot respectively. And finally the baculovirus particle were catched by transmission electron microscopy(TEM). Results PCR confirmed the identity of recombinant plasmid pENTR/D -TOPO -hKCTD9 and the recombined baculovirus containing hKCTD9 fusion gene. The laser confocal microscopy found that the hKCTD9 fusion proteins were expressed efficiently in infected sf9 cells and localized in the nuclei of sf9 cells. The expression hKCTD9 fusion protein was 55KD as shown by Western blot,other than 43KD in gene bank. TEM photograph showed that numerous recombinant baculocirus located in nuclei and cytoplasm in sf9 cells. Conclusion Human KCTD9 protein is expressed successfully using baculovirus expression system,which provides the basic tool for its further functional study.
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บทคดยอ ปจจบนการผลตนำยาตรวจหมโลหต anti-M และ anti-N ของฝายผลตนำยาแอนตซรมและผลตภณฑเซลล ศนยบรการโลหตแหงชาต สภากาชาดไทย ยงคงใชวธผลตจากนำเหลองกระตาย (rabbit polyclonal) ซงความแรงของแอนตบอดทผลตไดในแตละครงไมคงท เพราะตองทำการดดซบแอนตบอดชนดอนๆ ทไมตองการ (antibodies adsorption) การผลตโมโนโคลนลแอนตบอดดวยวธ murine monoclonal hybridoma technology จะไดแอนตบอดทมประสทธภาพสงกวาและสามารถผลตแอนตบอดชนดนนๆ ไดในปรมาณมากตามความตองการ รวมทงมความแรงและความจำเพาะมากกวา วตถประสงค เพอสรางเซลลสายพนธไฮบรโดมา (hybridoma cell line) ทสามารถนำมาผลตนำยาตรวจหมโลหต anti-M และ anti-N ดวยวธ murine monoclonal hybridoma technology วสดและวธการ ใชเซลลเมดเลอดขาวจากมามหน BAL B/c ทถกฉดกระตนดวย 20% เซลลเมดเลอดแดงของคนหม O phenotype M+N+ รวมทงสน 3 ครง ทำการเชอมเซลลเมดเลอดขาวทไดจากมามของหนทถกฉดกระตนแลวกบเซลลมะเรงของหนสายพนธเดยวกน (SP2/O) เมอเกดไฮบรโดมาจงนำนำเลยงเซลลไฮบรโดมาทดสอบกบ screening cells O 1 (M+N-) และ O 2 (M-N+) เพอคดเลอก hybridoma ทใหผลบวกกบ screening cells O 1 (M+N-) หรอ O 2 (M-N+) เพยงเซลลเดยวเทานนมาเลยงขยายตอ ทดสอบนำเลยงเซลลอกครงดวย identification panel cells และ papainized identification panel cells เพอคดเลอกโคลนทสราง anti-M หรอ anti-N แลวนำมาทำการเจอจางลดสดสวน (limiting dilution) เพอแยกโมโนโคลนและเลยงขยายโมโนโคลนทไดใหโตเตมท เกบแชแขงในไนโตรเจนเหลวเพอเกบไวเปนตนโคลน สวนนำเลยงเซลลเกบไวเพอทดสอบทาง serology อยางละเอยดตอไป ผลการศกษา งานวจยนสามารถสรางไฮบรโดมาทผลต anti-M จำนวน 1 โคลน คอ NBC-M2 (7H 3 2B 7 ) และไฮบรโดมาทผลต anti-N จำนวน 4 โคลน คอ NBC-N1(139G 3 G 2 ), NBC-N2 (9A 3 D 9 ), NBC-N3(9A 5 B 7 ) และ NBC-N4(9D 3 D 5 ) วจารณและสรป จากการทดสอบทาง serology เบองตนแสดงใหเหนวาไฮบรโดมาทสรางขนใหมทกโคลนสามารถนำไปผลตเปนนำยาตรวจหมโลหต anti-M และ anti-N ไดอยางไรกตามไฮบรโดมาทกโคลนเปนโคลนทสรางขนใหมยงตองทดสอบทาง serology อกมาก เพอใหเชอมนวา anti-M และ anti-N ทผลตไดใหมนมความเหมาะสมทจะนำไปผลตเปนนำยาตรวจหมโลหตไดอยางมประสทธภาพเพอทดแทนการผลตจากนำเหลองกระตาย
Polyclonal antibodies
Hybridoma technology
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Objective To obtain the fusion genes of several human orphan G protein coupled receptors (oGPCRs) with Gi1α subtype of G protein and their expression system.Methods The whole open reading frames of GPR45,GPR85,GPR174 and Gilα were cloned by RT-PCR from HepG2 cDNA separately,and the corresponding fusion genes were amplified by overlap extension PCR.Then,the fusion genes-containing pBacmids were successfully constructed with the Bac-to-Bac baculovirus expression system indicated by specific transposition and virus recombination.The insect Sf9 cells were transfected with pBacmid-oGPCRs-Gi1α,and the supernatant containing recombinant virus was harvested.With the supernatant,insect Sf9 cells were infected under an optimized condition (MOI=5,infection time=72 h) and the fusion proteins were prepared and detected by Western blotting.Results The three fusion genes of GPCR45,GPR85 or GPR174 with Gi1α were obtained.The corresponding fusion proteins could be properly prepared in Sf9 cells.Conclusion Human oGPCRs could be fused with Gilα,and the fusion genes could be expressed using the Bac-to-Bac baculovirus expression system in insect Sf9 cells.
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