[Objective] This study aimed to investigate the secretory expression of P97R1 gene of Mycoplasma hyopneumoniae（Mhp） in Pichia pastoris expression system and the primary application; of the expression product. [Metho...[Objective] This study aimed to investigate the secretory expression of P97R1 gene of Mycoplasma hyopneumoniae（Mhp） in Pichia pastoris expression system and the primary application; of the expression product. [Method] A pair of specific primers was designed to conduct PCR according to the Mhp P97R1 gene sequence in Genbank, and the amplified P97R1 gene was cloned into the pPICZa-A yeast expression vector to construct the secretory recombinant expression vector pPICZa-A-P97R1. The plasmid pPICZa-A-p97R1 linearized by Sac I was transformed into P. pastoris GSl15 by electroporation. Positive transformant identified by PCR was incubated to express P97R1 protein after methanol induction. And the expression product was identified using SDS-PAGE and Western-blotting anal.wsis. [Result] P97R1 protein was successfully expressed in the P. pastoris system, with a secre- tory amount of 499μg/ml, and revealed good reactogenicity. Meanwhile, an indirect ELISA method was established with P97R1 protein after the optimization of each reaction factor, which showed good specificity and repeatability according to repeated tests. [Conclusion] This study provides bases for developing the ELISA Kit for anti- body detection and genetically engineered vaccine to Mhp.展开更多
Blastomycosis, the systemic fungal disease of humans and animals caused by <i>Blastomyces dermatitidis </i>and the cryptic species <i>Blastomyces gilchristii</i><span>,<i> </i>...Blastomycosis, the systemic fungal disease of humans and animals caused by <i>Blastomyces dermatitidis </i>and the cryptic species <i>Blastomyces gilchristii</i><span>,<i> </i></span>is often misdiagnosed as a bacterial or viral pulmonary disease. Therefore, the development of improved immunodiagnostic assays for this disease has been the primary focus of research in our laboratory. The present study was designed to evaluate four <span>Blastomyces</span> yeast-phase lysate antigenic preparations (human, 597, Eagle River, WI;dog, ERC-2, WI;Human, B5927, Mountain Iron, MN;soil, 85, Georgia, ATCC 56920) for their ability to detect antibody in 48 serum specimens from dogs with diagnosed blastomycosis using an indirect ELISA (STD) compared to a biotin-streptavidin ELISA (B-SA). All four lysate antigens were able to detect antibod<span style="font-family:;" "="">ies</span><span style="font-family:;" "=""> in the specimens with mean absorbance values ranging from 0.930 (B5927) to 1.142 (ERC-2) with the STD ELSA and from 1.395 (B5927) to 1.775 (85) with the B-SA ELISA. The results indicated that both ELISA methods could be utilized for antibody detection, but the B-SA ELISA exhibited greater sensitivity than the STD ELISA with all four of the lysates.</span>展开更多
基金Supported by the National Natural Science Foundation of China(31100136)the Special Fund for Independent Innovation of Agricultural Science and Technology in Jiangsu Province[CX(12)5051]~~
文摘[Objective] This study aimed to investigate the secretory expression of P97R1 gene of Mycoplasma hyopneumoniae（Mhp） in Pichia pastoris expression system and the primary application; of the expression product. [Method] A pair of specific primers was designed to conduct PCR according to the Mhp P97R1 gene sequence in Genbank, and the amplified P97R1 gene was cloned into the pPICZa-A yeast expression vector to construct the secretory recombinant expression vector pPICZa-A-P97R1. The plasmid pPICZa-A-p97R1 linearized by Sac I was transformed into P. pastoris GSl15 by electroporation. Positive transformant identified by PCR was incubated to express P97R1 protein after methanol induction. And the expression product was identified using SDS-PAGE and Western-blotting anal.wsis. [Result] P97R1 protein was successfully expressed in the P. pastoris system, with a secre- tory amount of 499μg/ml, and revealed good reactogenicity. Meanwhile, an indirect ELISA method was established with P97R1 protein after the optimization of each reaction factor, which showed good specificity and repeatability according to repeated tests. [Conclusion] This study provides bases for developing the ELISA Kit for anti- body detection and genetically engineered vaccine to Mhp.
文摘Blastomycosis, the systemic fungal disease of humans and animals caused by <i>Blastomyces dermatitidis </i>and the cryptic species <i>Blastomyces gilchristii</i><span>,<i> </i></span>is often misdiagnosed as a bacterial or viral pulmonary disease. Therefore, the development of improved immunodiagnostic assays for this disease has been the primary focus of research in our laboratory. The present study was designed to evaluate four <span>Blastomyces</span> yeast-phase lysate antigenic preparations (human, 597, Eagle River, WI;dog, ERC-2, WI;Human, B5927, Mountain Iron, MN;soil, 85, Georgia, ATCC 56920) for their ability to detect antibody in 48 serum specimens from dogs with diagnosed blastomycosis using an indirect ELISA (STD) compared to a biotin-streptavidin ELISA (B-SA). All four lysate antigens were able to detect antibod<span style="font-family:;" "="">ies</span><span style="font-family:;" "=""> in the specimens with mean absorbance values ranging from 0.930 (B5927) to 1.142 (ERC-2) with the STD ELSA and from 1.395 (B5927) to 1.775 (85) with the B-SA ELISA. The results indicated that both ELISA methods could be utilized for antibody detection, but the B-SA ELISA exhibited greater sensitivity than the STD ELISA with all four of the lysates.</span>
