Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (7): 269-277.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1299
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WANG Guang-li1,2(), FAN Chan1,2, WANG Hui1,2, LU Hui-fang1,2, XIA Ling-yin1,2, HUANG Jian1,2, MIN Xun1,2()
Received:
2021-10-14
Online:
2022-07-26
Published:
2022-08-09
Contact:
MIN Xun
E-mail:1947952722@qq.com;zmchj2001@163.com
WANG Guang-li, FAN Chan, WANG Hui, LU Hui-fang, XIA Ling-yin, HUANG Jian, MIN Xun. Prokaryotic Expression,Purification,Identification,and Polyclonal Antibody Preparation of Vibrio cholerae Hemolysin HlyA[J]. Biotechnology Bulletin, 2022, 38(7): 269-277.
Fig.1 Construction of recombinant plasmids pET28a-hlyA,pET32 a-hlyA and pCold TF-hlyA. M:DNA marker DL5000 bp;A:electrophoresis results of hlyA gene PCR amplification(1:hlyA fragment). B-D:Electrophoresis results of pET28a-hlyA,pET32a-hlyA and pCold TF-hlyArecombinant plasmid bacterial liquid PCR,respectively(1:positive control;2-4:positive cloning bacteria)
Fig.2 SDS-PAGE analysis of pET28a-hlyA expression pro-ducts in E. coli BL21(DE3)induced with IPTG M:180 kD protein marker;A:1:uninduced E. coli BL21(DE3)with recombinant plasmid pET28a-hlyA;2-3:induced E. coli BL21(DE3)with recombinant plasmid pET28a-hlyA at 10℃,120 r/min by 0.05 mmol/L and 0.1 mmol/L IPTG for 16 h;4:induced E. coli BL21(DE3)with recombinant plasmid pET28a-hlyA at 15℃,100 r/min by 0.05 mmol/L IPTG for 12 h;5 and 7:lysate pellet of induced bacteria;6 and 8:lysate supernatant of induced bacteria. B:1:uninduced E. coli BL21(DE3)with recombinant plasmid pET28a-hlyA;2:lysate pellet of induced bacteria at 23℃ by 0.05 mmol/L IPTG for 10 h;3:lysate supernatant of induced bacteria at 23℃
Fig.3 SDS-PAGE analysis of pET32a-hlyA expression pr-oducts in E. coli BL21(DE3)induced with IPTG M:250 kD protein marker. A:1:uninduced E. coli BL21(DE3)with recombinant plasmid pET32a-hlyA;2-3:induced E. coli BL21(DE3)with recombinant plasmid pET32a-hlyA at 15℃,100 r/min by 0.05 and 0.1 mmol/L IPTG for 16 h;4 and 5:induced E. coli BL21(DE3)with recombinant plasmid pET32a-hlyA at 25℃,120 r/min by 0.05 and 0.1 mmol/L IPTG for 10 h;6-9:lysate supernatant of induced bacteria;10-13:lysate pellet of induced bacteria. B:1:uninduced E. coli BL21(DE3)with recombinant plasmid pET32a-hlyA;2 and 5:induced E. coliBL21(DE3)with recombinant plasmid pET32a-hlyA at 20℃,100 r/min by 0.05 and 0.1 mmol/L IPTG for 12 h;3 and 6:lysate pellet of induced bacteria;4 and 7:lysate supernatant of induced bacteria
Fig.4 SDS-PAGE analysis of pCold TF-hlyA expression products in E. coli BL21 induced with IPTG and the purified HlyA protein M:250 kD protein marker;1:uninduced E. coli BL21 with recombinant plasmid pCold TF -hlyA;2:induced E. coli BL21 with recombinant plasmid pCold TF -hlyA;3:lysate supernatant of induced bacteria;4-9:1×Washing Buffer contain of 10,20,30,40,300,500 mmol/L imidazole through Ni-chelating affinity chromatography
Fig.5 Hemolytic activity determination of recombinant expressive protein HlyA 1:Positive control(1% Trion-X);2:negative control(PBS);3:0 μg HlyA protein incubateed with 2% rabbit red blood cells;4:4 μg HlyA protein incubateed with 2% rabbit red blood cells;5:8 μg HlyA protein co-incubated with 2% rabbit red blood cells
Fig.8 Mass spectrometric profiles of HlyA protein A mass spectrum of the peptide sequence SASFTVDWDHPVFTGGRPVNLQLAS-FNNR that matches the amino acid sequence of the HlyA protein is shown
Fig. 9 Detection of Anti-HlyA polyclonal antibody neutrali-zation activity A:Analysis of the inhibitory effect of anti-HlyA polyclonal antibody on the hemolytic activity of HN375△tagH strain;B:Analysis of the inhibitory effect of anti-HlyA polyclonal antibody on the hemolytic activity of HN375 strain and two clinical strains. *** indicates that there is a significant difference in the data between two groups(P < 0.001)
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