Secretory anti-gp96 scFv fragment was expressed in Pichia pastoris to obtain a small molecule antibody that specifically recognizes heat shock protein gp96. The gp96-scFv fragment gene was synthesized and cloned to Pichia pastoris expression plasmid pPICZa-A. Pichia pastoris X33 was electroporated with the linearized recombinant expression vector, and expression of gp96-scFv fragment was induced by methanol. The His-tagged recombinant protein was then purified by affinity chromatography and analyzed with SDS-PAGE and Western blotting assays. The biological activities of recombinant gp96-scFv fragment were determined by Western blotting, Immunofluorescence, ELISA and FACS assays. The gp96-scFv fragment was expressed successfully in Pichia pastoris. About 50 mg of recombinant protein could be purified from 1 liter of the Pichia pastoris culture supernatant. Its molecular weight was about 15 kDa. The gp96-scFv fragment could specifically bind to gp96 protein by Western blotting, immunofluorescence, ELISA and FACS analyses. Pichia pastoris-expressed gp96-scFv fragment specifically recognizes gp96 protein, which could be used for Western blotting, Immunofluorescence, ELISA and FACS analyses.
Blotting, Western ; Chromatography, Affinity ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Membrane Glycoproteins ; immunology ; Pichia ; metabolism ; Plasmids ; Recombinant Proteins ; biosynthesis ; Single-Chain Antibodies ; biosynthesis

Objective To investigate the relationship between high-sensitivity C-reactive protein (hsCRP) and retinal vascular diameter.Methods This study was a cross-sectional study,the population was from the Kailuan study (clinical trial research registration NO.:ChiCTR-TNC-1100 1489) in 2012-2013 (age ≥ 40 years).All the people was checked by cardiovascular and epidemiological examination,of which 3 447 people had an analytical results of fundus color photography.The retinal vascular diameter was analyzed by software.Take the value of hsCRP 1％ to 99％ points and 3 363 people were finally included in the statistical analysis.The mean age of 3 363 people (male 1 880,female 1 483) was 56.69± 10.10 years old.The correlation between hsCRP and central retinal artery equivalent (CRAE),central retinal vein equivalent (CRVE),arteriovenous ratio (AVR) were evaluated by Spearman correlation coefficient.Multivariate linear regression analysis was used to futher analyze the association between hsCRP and retinal fundus diameter parameters.Results The median value of hsCRP was 1.10 (0.50,2.21) mmo/L,CRAE was (174.16±24.36) μm,CRVE was (282.70±26.32) μm,AVR was 0.62-±0.09.Spearman correlation analysis showed that hsCRP was positively correlated with CRVE (r =0.052,P =0.003) and negatively correlated with AVR (r =-0.052,P =0.002).The non-normal variables hsCRP were transformed into continuous variables lghsCRP,and CRAE,CRVE and AVR were used for multivariate linear regression analysis.After a number of factors were gradually corrected,the results showed that association between lghsCRP and CRVE (β =3.428) or AVR (β =-0.006) was statistically significant (P＜0.05).LghsCRP was a risk factor for CRVE both in men and women after the calculation of gender stratification.Conclusions CRVE increased and AVR decreased with the increase of hsCRP.The changes of retinal vascular diameter may be associated with hsCRP as a marker to predict cardiovascular and cerebrovascular disease.

With the progression of research on extrachromosomal DNA(ecDNA),it has been shown that ecDNA exists mainly in tumor cells and plays a crucial role in tumor heterogeneity and drug resistance.ecDNA is observed in several cancer types,but rarely in normal cells.Due to their strong oncogene amplification and dynamic alteration capabilities,patients with ecDNA-containing tumor cells often have negative clinical prognoses.Research has confirmed the presence of ecDNA in the cancer cells of patients with small cell lung cancer(SCLC).This re-view provides a comprehensive summary of the formation mechanism of ecDNA,the processes through which it is amplified in cancer cells,the mechanisms through which ecDNA promotes tumor growth,recurrence,and metastasis,and its relationship with high drug resistance in SCLC.Finally,we generalize the treatment direction for ecDNA-enriched SCLCs,thereby guiding future research.