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Prohibitin as a novel autoantigen in rheumatoid arthritis.

Shi LL, Chen P, Xun YP, Yang WK, Yang CH, Chen GY, Du HW - Cent Eur J Immunol (2015)

Bottom Line: First, recombinant human prohibitin (rhPHB) protein was cloned, expressed, and purified.Reactivity of serum IgG against rhPHB was detected in 26 of 86 RA patients (30.3%), 7 of 86 systemic lupus erythematosus (SLE) patients (8.1%), and 1 of 86 apparently healthy donors (HC) (1.2%).Prohibitin was proved to be a novel autoantigen and the corresponding anti-prohibitin autoantibodies were present in the RA patients' blood circulation.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry & Biotechnology Engineering, University of Science & Technology Beijing, Beijing, China.

ABSTRACT

The aim of this study: The aim of this study was to verify whether prohibitin is a novel autoantigen in rheumatoid arthritis.

Material and methods: First, recombinant human prohibitin (rhPHB) protein was cloned, expressed, and purified. Then the anti-prohibitin autoantibodies were detected by western blotting by using rhPHB protein to incubate sera from patients with rheumatoid arthritis (RA). Next, immunoprecipitation was employed to further illustrate whether anti-prohibitin antibodies exist in RA patients. And finally, autoantibodies against the rhPHB protein were investigated using a homemade ELISA kit through the assessment of 258 real clinical samples.

Results: It was revealed that anti-prohibitin antibodies existed in the sera of patients with RA. Reactivity of serum IgG against rhPHB was detected in 26 of 86 RA patients (30.3%), 7 of 86 systemic lupus erythematosus (SLE) patients (8.1%), and 1 of 86 apparently healthy donors (HC) (1.2%).

Conclusions: Prohibitin was proved to be a novel autoantigen and the corresponding anti-prohibitin autoantibodies were present in the RA patients' blood circulation.

No MeSH data available.


Related in: MedlinePlus

Expression of prohibitin. A) PCR of the prohibitin gene. Agarose gels showing the amplified 819-bp fragment. B) Sodium dodecyl sulphate-poly-acrylamide gel electrophoresis (SDS-PAGE) analysis of prohibitin protein. M, protein marker; lane 1, cell extracts of pET-28a(+)-prohibitin/BL21 after isopropyl-β-D-thiogalactopyranoside (IPTG) induction for 6 h at 37°C; lane 2, cell extracts of pET28a (+)/BL21 after IPTG induction. C) Prohibitin was identified by mass spectrometry
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Figure 0001: Expression of prohibitin. A) PCR of the prohibitin gene. Agarose gels showing the amplified 819-bp fragment. B) Sodium dodecyl sulphate-poly-acrylamide gel electrophoresis (SDS-PAGE) analysis of prohibitin protein. M, protein marker; lane 1, cell extracts of pET-28a(+)-prohibitin/BL21 after isopropyl-β-D-thiogalactopyranoside (IPTG) induction for 6 h at 37°C; lane 2, cell extracts of pET28a (+)/BL21 after IPTG induction. C) Prohibitin was identified by mass spectrometry

Mentions: The PCR products showed a single band at the position of 819 bp as indicated on 1% agarose gel (Fig. 1A). The extracted band was excised by EcoR I and Hind III and then ligated into the pET-28a (+) vector, which had been digested with EcoR I and Hind III, to form the recombinant plasmid. The construct was transformed in E. coli BL21 (DE). After six hours of induction by isopropyl-P-D-thio-A galactopyranoside (IPTG), the recombination cells were lysed ultrasonically and the cell extracts were analysed by SDS-PAGE. High levels of recombinant prohibitin were observed (Fig. 1B). The protein was analysed by means of LC-MALDI-TOF/TOF (Fig. 1C).


Prohibitin as a novel autoantigen in rheumatoid arthritis.

Shi LL, Chen P, Xun YP, Yang WK, Yang CH, Chen GY, Du HW - Cent Eur J Immunol (2015)

Expression of prohibitin. A) PCR of the prohibitin gene. Agarose gels showing the amplified 819-bp fragment. B) Sodium dodecyl sulphate-poly-acrylamide gel electrophoresis (SDS-PAGE) analysis of prohibitin protein. M, protein marker; lane 1, cell extracts of pET-28a(+)-prohibitin/BL21 after isopropyl-β-D-thiogalactopyranoside (IPTG) induction for 6 h at 37°C; lane 2, cell extracts of pET28a (+)/BL21 after IPTG induction. C) Prohibitin was identified by mass spectrometry
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4472543&req=5

Figure 0001: Expression of prohibitin. A) PCR of the prohibitin gene. Agarose gels showing the amplified 819-bp fragment. B) Sodium dodecyl sulphate-poly-acrylamide gel electrophoresis (SDS-PAGE) analysis of prohibitin protein. M, protein marker; lane 1, cell extracts of pET-28a(+)-prohibitin/BL21 after isopropyl-β-D-thiogalactopyranoside (IPTG) induction for 6 h at 37°C; lane 2, cell extracts of pET28a (+)/BL21 after IPTG induction. C) Prohibitin was identified by mass spectrometry
Mentions: The PCR products showed a single band at the position of 819 bp as indicated on 1% agarose gel (Fig. 1A). The extracted band was excised by EcoR I and Hind III and then ligated into the pET-28a (+) vector, which had been digested with EcoR I and Hind III, to form the recombinant plasmid. The construct was transformed in E. coli BL21 (DE). After six hours of induction by isopropyl-P-D-thio-A galactopyranoside (IPTG), the recombination cells were lysed ultrasonically and the cell extracts were analysed by SDS-PAGE. High levels of recombinant prohibitin were observed (Fig. 1B). The protein was analysed by means of LC-MALDI-TOF/TOF (Fig. 1C).

Bottom Line: First, recombinant human prohibitin (rhPHB) protein was cloned, expressed, and purified.Reactivity of serum IgG against rhPHB was detected in 26 of 86 RA patients (30.3%), 7 of 86 systemic lupus erythematosus (SLE) patients (8.1%), and 1 of 86 apparently healthy donors (HC) (1.2%).Prohibitin was proved to be a novel autoantigen and the corresponding anti-prohibitin autoantibodies were present in the RA patients' blood circulation.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry & Biotechnology Engineering, University of Science & Technology Beijing, Beijing, China.

ABSTRACT

The aim of this study: The aim of this study was to verify whether prohibitin is a novel autoantigen in rheumatoid arthritis.

Material and methods: First, recombinant human prohibitin (rhPHB) protein was cloned, expressed, and purified. Then the anti-prohibitin autoantibodies were detected by western blotting by using rhPHB protein to incubate sera from patients with rheumatoid arthritis (RA). Next, immunoprecipitation was employed to further illustrate whether anti-prohibitin antibodies exist in RA patients. And finally, autoantibodies against the rhPHB protein were investigated using a homemade ELISA kit through the assessment of 258 real clinical samples.

Results: It was revealed that anti-prohibitin antibodies existed in the sera of patients with RA. Reactivity of serum IgG against rhPHB was detected in 26 of 86 RA patients (30.3%), 7 of 86 systemic lupus erythematosus (SLE) patients (8.1%), and 1 of 86 apparently healthy donors (HC) (1.2%).

Conclusions: Prohibitin was proved to be a novel autoantigen and the corresponding anti-prohibitin autoantibodies were present in the RA patients' blood circulation.

No MeSH data available.


Related in: MedlinePlus