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Objective:To observe the inhibition of SARS-CoV-2 spike protein (S-protein) on the proliferation of human retinal pigment epithelium (RPE) cells.Methods:SARS-CoV-2 S-protein gene fragment expression plasmid (p3xflag-S) was constructed and transfected into human RPE, HEK293 cells. DNA sequencing was used for identification, and the expression of Flag-S was detected by Western blot. HEK293 cells were divided into the cells 1, 2, 3 and 4 and transfected with GFP11 plasmid and vector, GFP1-10 plasmid and vector, transfected with GFP11 and pCMV-HA-ACE2 plasmid, GFP1-10 and p3xflag-S plasmid. Cell 1 was co-cultured with cell 2 (control group 1), cell 2 with cell 3 (control group 2), cell 3 with cell 4 (observation group), and cell 1 mixed with cells 2, 3 and 4 (control group 3). Bright-field microscopy and fluorescence microscopy were used to observe cell fusion. RPE cells were divided into control group and overexpression S-protein group. The cell cycle was detected by flow cytometry; the cell proliferation level was detected by Counting Kit 8 (CCK-8); and the S-protein expression level in RPE cells was detected by Western blot. The Student’s t-test was performed for comparison between groups. Results:DNA sequence assay showed that S-protein cDNA was fused with flag-tagged protein. Western blot assay showed that S-protein-related expression was elevated in transfected HEK293 cells compared with untransfected p3xflag-S cells. Large, multinucleated fused cell clusters were visible under bright-field microscopy; multiple nuclear with distinct green fluorescence were visible in the fused cells under fluorescence microscopy. Western blot assay showed elevated S-protein-related expression in transfected p3xflag-S plasmid RPE cells compared to untransfected p3xflag-S plasmid RPE cells. CCK-8 results showed that the proliferative capacity of RPE cells in the S-protein overexpression group was significantly reduced compared with the control group, with statistically significant differences ( t=22.70, 16.75, 23.38; P<0.000 1). The results of flow cytometry showed that the G1 phase cells in the control and overexpression S-protein groups were 41.1 % and 67.0%, respectively; compared with the control group, the G1 phase cells in the overexpression S-protein group were significantly higher, and the difference was statistically significant ( t=4.76, P=0.018). The apoptosis rate was significantly increased in the S-protein overexpression group compared with the control group, and the difference was statistically significant ( t=4.91, P=0.008). Conclusion:Overexpression of the SARS-CoV-2 spike protein reduced the proliferation of human RPE cells.
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Objective:To express and purify the fusion protein of GST-FILIP-1L and prepare its polyclonal antibody. Methods:The constructed recombinant expression vectors pGEX-4T3-FILIP-1L were transformed into Escherichia Coli BL21. FILIP-1L fusion protein was induced by IPTG and purified by Glutathion Sepharse 4B . The rabbit was immunized by the purified fusion protein,and pro-duced serum with anti-FILIP-1L antibody. The titer of polyclonal antibody was detected by ELISA, the anti-FILIP-1L polyclonal antibody was purified by Active and its combining specificity with FILIP-1L protein was further identified by Western blot. Results:The GST-FILIP-1L fusion protein was highly expressed in E. coli, and its specific polyclonal antibody was obtained after the immunization. The polyclonal antibody purified by Active Ester Agrose was able to combine specially with FILIP-1L protein and transformed FILIP-1L protein in 293 cells and FILIP-1L protein of liver cancer cells, respectively. Conclusion: The GST-FILIP-1L fusion protein was expressed successfully,the anti-GST-FILIP-1L polyclonal antibodies with high titer and specificity are successfully prepared,these antibodies provide an useful experimental tool to research the biological function of the FILIP-1L protein.
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AIM:To clarify the impact of heart shock factor 4b (Hsf4b) K65R mutation on the regulation of downstream protein expression .METHODS:Non-functional Lys mutant plasmid pWZL-blast-HA-Hsf4b/K65R was genera-ted by replacing single , homologous amino acids using KOD-Plus-Mutagenesis-Kit.Mouse lens epithelial mLEC stable cell lines expressing Hsf4b or Hsf4b/K65R were constructed by lentivirus infection .The expression of Hsf4b in the mutant and the wildtype mLEC cells was confirmed by Western blotting .The expression of Hsf4b downstream proteins such as heat shock protein ( Hsp)70, Hsp90, Hsp27 and CryAB was examined by Western blotting and real-time PCR.RESULTS:The results of PCR and DNA sequencing confirmed the successful construction of mLEC Hsf 4b/K65R mutant.The K65R mutation didn’t influence Hsf4b expression in the mLEC cells.After K65R mutation in Hsf4b, the expression levels of Hsp27 and CryAB were down-regulated and the expression of Hsp 70i and Hsp90a upregulated.CONCLUSION: pWZL-blast-HA-Hsf4b/K65R can be used to construct a stable cell line by infecting with lentivirus .Hsf4b/K65R mutation influ-ences the regulation of downstream heat shock proteins .
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ObjectiveTo investigate the expression and clinical significance of heat shock transcription factor 1 (HSF1) protein in human hepatocellular carcinoma (HCC) tissues,and deduce the probable molecular mechanism of HSF1 in the development and advancement of HCC.MethodsSixty-seven samples of HCC tissue and 21 samples of normal liver tissue were obtained from March 2006 to March 2007 at the Xijing Hospital.The expressions of HSF1 protein and heat shock protein 70 (HSP70) were detected by using immunohistochemistry.The probable molecular mechanism of HSF1 in the development and advancement of HCC was deduced according to the relationship between the expressions of HSF1 protein and HSP70.Positive rates of HSF1 protein in different tissues and the relationship between HSF1 protein expression in the HCC tissues and clinical pathological factors were analyzed by the chi-square test and by calculating Fisher exact probability,respectively.The correlation between the expressions of HSF1 protein and HSP70 in the HCC tissue was analyzed by the Spearman correlation coefficient.The survival curve was drawn by the Kaplan-Meier method,and the survival rate was analyzed by the Log-rank test.ResultsThe positive rates of HSF1 protein expression was 69％ (46/67) in the HCC tissue,which was significantly higher than 29％ (6/21) in the normal liver tissue ( x2 =10.628,P ＜ 0.05 ),The positive rates of HSP70 expression in the HCC tissue was 57％ (38/67),which was significantly higher than 24％ (5/21) in the normal liver tissue ( x2 =6.929,P ＜ 0.05 ).The expression of HSF1 protein in the HCC tissue was positively correlated with that of HSP70 (r=0.319,P ＜0.05).The high expression of HSF1 protein was correlated with the integrity of capsule of HCC,tumor differentiation and TNM stage (x2 =5.935,9.762,5.159,11.267,P＜0.05 ),while the high expression of HSF1 protein was not correlated with the gender,age,levels of hepatitis B surface antigen and alpha fetoprotein,and portal vein tumor thrombus ( x2 =0.822,0.172,2.059,P ＞0.05 ).The survival time was (21.4 ± 1.9 )months for patients with positive HSF1 protein expression and (29.8 ± 2.7 ) months for patients with negative HSF1 protein expression.There was a significant difference in the survival time between patients with positive and negative HSF1 protein expression ( x2 =4.276,P ＜ 0.05 ).Conclusions HSF1 is correlated with the development,advancement,invasion,metastasis and malignant prognosis of HCC.HSF1 takes effects by regulating the expression of HSP70,and it has a good perspective of clinical application for the diagnosis and treatment of HCC.