Biotechnology Bulletin ›› 2024, Vol. 40 ›› Issue (5): 310-320.doi: 10.13560/j.cnki.biotech.bull.1985.2023-1114
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WANG Xin-xin1,2(
), GUAN Yu-zhu1,2, LI Xiao-wei1,2, HONG Wei1,3, WU Dao-yan1,2, KANG Ying-qian1,2, LIU Yong-chang1,2, CHEN Zheng-hong1,2(), CUI Gu-zhen1,2()
Received:2023-11-27
Online:2024-05-26
Published:2024-03-28
Contact:
CHEN Zheng-hong, CUI Gu-zhen
E-mail:wxx_999w@163.com;chenzhenghong@gmc.edu.cn;cuiguzhen@gmc.edu.cn
WANG Xin-xin, GUAN Yu-zhu, LI Xiao-wei, HONG Wei, WU Dao-yan, KANG Ying-qian, LIU Yong-chang, CHEN Zheng-hong, CUI Gu-zhen. Function of katA in Helicobacter pylori and Its Role in the Tolerance to Oxidative Damage[J]. Biotechnology Bulletin, 2024, 40(5): 310-320.
| 菌株及质粒Strain and plasmid | 相关特征Relevant characteristics | 来源Source |
|---|---|---|
| Strains E.coli | ||
| DH5α | Clone strain, F-lacZ ΔM15Δ（lacZYA-aragF）relA1 | This lab |
| DH5α::pSD | Derived fom DH5α, carrying plasmid pSD, kanR | This lab |
| DH5α:: pSD-KO-katA | Derived fom DH5α, carrying plasmid pSD-KO-katA, kanR | This work |
| DH5α:: pCD | Derived fom DH5α, carrying plasmid pCD, CmR | This work |
| DH5α:: pCD-EX-katA | Derived fom DH5α, carrying plasmid pCD-EX-katA, CmR | This work |
| DH5α:: pET-28a-katA | Derived fom DH5α, carrying plasmid pET-28a-katA, kanR | This work |
| BL21（DE3） | Expression strain, F-lon-11Δ（ompT-nfrA）885Δ（galM-ybhJ）884 | This lab |
| BL21（DE3）::pET-28a | Derived fom BL21（DE3）, carrying plasmid pET-28a, kanR | This work |
| BL21（DE3）::pET-28a-katA | Derived fom BL21（DE3）, carrying plasmid pET-28a-katA, kanR | This work |
| H.pylori | ||
| HpG272 | Clinical strains | This lab |
| Hp∆katA | H.pylori strain G272, knockout gene katA | This work |
| Hp∆katA::katA | H.pylori strain HpΔkatA::katA, anaplerosis gene katA | This work |
| Plasmids | ||
| pSD | From pill570, suicide plasmid, kanR | This lab |
| PCD | From pSD, suicide plasmid, CmR | This lab |
| pSD-KO-katA | Suicide plasmid, knockout gene katA, kanR | This work |
| pCD-EX-katA | HpΔkatA::katA replenishing plasmid, CmR | This work |
| pET-28a | N-T7, N-His, C-His, E. coli protein expression vector | This lab |
| pET-28a-katA | Carrying katA gene, E. coli protein expression vector | This work |
Table 1 Strains and plasmids used in this study
| 菌株及质粒Strain and plasmid | 相关特征Relevant characteristics | 来源Source |
|---|---|---|
| Strains E.coli | ||
| DH5α | Clone strain, F-lacZ ΔM15Δ（lacZYA-aragF）relA1 | This lab |
| DH5α::pSD | Derived fom DH5α, carrying plasmid pSD, kanR | This lab |
| DH5α:: pSD-KO-katA | Derived fom DH5α, carrying plasmid pSD-KO-katA, kanR | This work |
| DH5α:: pCD | Derived fom DH5α, carrying plasmid pCD, CmR | This work |
| DH5α:: pCD-EX-katA | Derived fom DH5α, carrying plasmid pCD-EX-katA, CmR | This work |
| DH5α:: pET-28a-katA | Derived fom DH5α, carrying plasmid pET-28a-katA, kanR | This work |
| BL21（DE3） | Expression strain, F-lon-11Δ（ompT-nfrA）885Δ（galM-ybhJ）884 | This lab |
| BL21（DE3）::pET-28a | Derived fom BL21（DE3）, carrying plasmid pET-28a, kanR | This work |
| BL21（DE3）::pET-28a-katA | Derived fom BL21（DE3）, carrying plasmid pET-28a-katA, kanR | This work |
| H.pylori | ||
| HpG272 | Clinical strains | This lab |
| Hp∆katA | H.pylori strain G272, knockout gene katA | This work |
| Hp∆katA::katA | H.pylori strain HpΔkatA::katA, anaplerosis gene katA | This work |
| Plasmids | ||
| pSD | From pill570, suicide plasmid, kanR | This lab |
| PCD | From pSD, suicide plasmid, CmR | This lab |
| pSD-KO-katA | Suicide plasmid, knockout gene katA, kanR | This work |
| pCD-EX-katA | HpΔkatA::katA replenishing plasmid, CmR | This work |
| pET-28a | N-T7, N-His, C-His, E. coli protein expression vector | This lab |
| pET-28a-katA | Carrying katA gene, E. coli protein expression vector | This work |
| 引物Primer | 序列Sequence（5'-3'） | 注释Notes |
|---|---|---|
| Kan-1 | GAATTCGAGCTCGGTACCCG | kanR 基因扩增和检测引物 |
| Kan-2 | CCCGGGTCATTATTCCCTCC | |
| Kat-1 | CTGCAGGGTATAAAAACTCACCGCCCCA | 敲除质粒及敲除菌株检测引物 |
| Kat-2 | ATCGATACCTAGTTTCAAGCCTTGCA | |
| KatA-1 | ATCGACTGCAGGGTATAAAAACTCACCGCCCCA | katA 基因敲除载体上游同源臂扩增 |
| KatA-2 | ATCGAGAATTCCGTAATTGACACTAAGCCGATTAC | |
| KatA-3 | ATCGAGGATCCCTTATTTTTTAGGAACGCTTTGTC | katA 基因敲除载体下游同源臂扩增 |
| KatA-4 | ATCGAATCGATACCTAGTTTCAAGCCTTGCA | |
| Chl-1 | TTAAAAAAATTACGCCCCGCCCTG | CmR 基因扩增和检测引物 |
| Chl-2 | AAATGGAGAAAAAAATCACTGGATATACCACCG | |
| KatA -5 | CCCCGGGGACCTGCAGGCCGCTTGCACGGGT | katA 基因回补载体上游同源臂 |
| KatA -6 | TTTTTTAAGGCAGTCTGCAGGCGACCAAAACCTCTCATGGAG | |
| KatA -7 | TTGACAGCTTATCATCGATGTGAGTCAGAATGTCTTTCACAACCC | katA 基因回补载体下游同源臂 |
| KatA -8 | CTCCTGAAAATCTCGGATCCGGGCGGTTTCACTGAGAAAACTT | |
| KatA -9 | CAGTGAAACCGCCCGGATCTTTATAAATTCTAAAGGGG | katA 基因回补载体Hp尿素酶启动子PureA 扩增 |
| KatA -10 | TTAGCTCCTGAAAATCTCGGATCCTTATTCTCCTATTCTTAAAGTG | |
| KatA -11 | AACACTTTAAGAATAGGAGAATAAGATGGTTAATAAAGATGTGAAACAAACTACTGC | 回补载体 katA 基因扩增 |
| KatA -12 | TTAGCTCCTGAAAATCTCGTTACTTTTTCTTTTTTGTGTGGTGCATGTCT | |
| Kat-3 | TTGACAGCTTATCATCGATGTGAGTCAGAATGTCTTTCACAACCC | 回补质粒及回补菌株检测引物 |
| Kat-4 | CCCCGGGGACCTGCAGGCCGCTTGCACGGGT | |
| KatA-R | GTCGACGGAGCTCGAATTCGTTACTTTTTCTTTTTTGTGTGGT | katA 基因扩增 |
| KatA-F | AGCAAATGGGTCGCGGATCATGGTTAATAAAGATGTGAAAC | |
| KatA-C1 | CAAAAAACCCCTCAAGACCCGTTTAG | KatAG272 蛋白表达质粒检测引物 |
| KatA-C2 | ACCGGCATACTCTGCGACATC |
Table 2 Primers used in this study
| 引物Primer | 序列Sequence（5'-3'） | 注释Notes |
|---|---|---|
| Kan-1 | GAATTCGAGCTCGGTACCCG | kanR 基因扩增和检测引物 |
| Kan-2 | CCCGGGTCATTATTCCCTCC | |
| Kat-1 | CTGCAGGGTATAAAAACTCACCGCCCCA | 敲除质粒及敲除菌株检测引物 |
| Kat-2 | ATCGATACCTAGTTTCAAGCCTTGCA | |
| KatA-1 | ATCGACTGCAGGGTATAAAAACTCACCGCCCCA | katA 基因敲除载体上游同源臂扩增 |
| KatA-2 | ATCGAGAATTCCGTAATTGACACTAAGCCGATTAC | |
| KatA-3 | ATCGAGGATCCCTTATTTTTTAGGAACGCTTTGTC | katA 基因敲除载体下游同源臂扩增 |
| KatA-4 | ATCGAATCGATACCTAGTTTCAAGCCTTGCA | |
| Chl-1 | TTAAAAAAATTACGCCCCGCCCTG | CmR 基因扩增和检测引物 |
| Chl-2 | AAATGGAGAAAAAAATCACTGGATATACCACCG | |
| KatA -5 | CCCCGGGGACCTGCAGGCCGCTTGCACGGGT | katA 基因回补载体上游同源臂 |
| KatA -6 | TTTTTTAAGGCAGTCTGCAGGCGACCAAAACCTCTCATGGAG | |
| KatA -7 | TTGACAGCTTATCATCGATGTGAGTCAGAATGTCTTTCACAACCC | katA 基因回补载体下游同源臂 |
| KatA -8 | CTCCTGAAAATCTCGGATCCGGGCGGTTTCACTGAGAAAACTT | |
| KatA -9 | CAGTGAAACCGCCCGGATCTTTATAAATTCTAAAGGGG | katA 基因回补载体Hp尿素酶启动子PureA 扩增 |
| KatA -10 | TTAGCTCCTGAAAATCTCGGATCCTTATTCTCCTATTCTTAAAGTG | |
| KatA -11 | AACACTTTAAGAATAGGAGAATAAGATGGTTAATAAAGATGTGAAACAAACTACTGC | 回补载体 katA 基因扩增 |
| KatA -12 | TTAGCTCCTGAAAATCTCGTTACTTTTTCTTTTTTGTGTGGTGCATGTCT | |
| Kat-3 | TTGACAGCTTATCATCGATGTGAGTCAGAATGTCTTTCACAACCC | 回补质粒及回补菌株检测引物 |
| Kat-4 | CCCCGGGGACCTGCAGGCCGCTTGCACGGGT | |
| KatA-R | GTCGACGGAGCTCGAATTCGTTACTTTTTCTTTTTTGTGTGGT | katA 基因扩增 |
| KatA-F | AGCAAATGGGTCGCGGATCATGGTTAATAAAGATGTGAAAC | |
| KatA-C1 | CAAAAAACCCCTCAAGACCCGTTTAG | KatAG272 蛋白表达质粒检测引物 |
| KatA-C2 | ACCGGCATACTCTGCGACATC |
Fig. 1 Conservative analysis of amino acid sequence for katA Red: Conservative amino acids. Yellow: Relatively conserved amino acid. Colorless: Non conserved amino acids
Fig. 2 PCR amplification for katA and construction of expression vector A: PCR amplification for katA gene. B: Expression plasmid PCR identification (Lane 1-2: PCR identification for positive plasmid; lane 3: blank control, primers used in this identification are KatA-C1/KatA-C2). C: pET28a-katA expression plasmid
Fig. 3 Protein expression, isolation and purification for KatAG272 A: The induced expression of KatAG272 protein in Escherichia coli（Lane 1, 4, 7, 10: Bacterial precipitation before ultrasound fragmentation; lane 2, 5, 8, 11: culture supernatant before ultrasound fragmentation; lane 3, 6, 9, 12: supernatant after ultrasound fragmentation; lane 13-14: control of bacterial precipitation before wild-type ultrasound fragmentation containing pET28a vector）. B: KatAG272 protein isolation and purification（Lane 1: Flow through fluid; lane 2:10 mmol/L imidazole rinse; lane 3-5: 200 mmol/L imidazole elution）
Fig. 4 Analysis and identification of catalase activity A: Detection of catalase activity of KatAG272 pure enzyme. B: Detection of KatAG272 catalase activity in recombinant E. coli; C: The effect of temperature on the activity of KatAG272. D: The effect of pH on the activity of KatAG272. E: KatAG272 protein temperature tolerance. F: KatAG272 protein pH tolerance
Fig. 5 Construction and identification for katA-knockout strain HpΔkatA A: Schematics of HpΔkatA construction. B: Identification of HpΔkatA（Lane 1: primer Kan-1 and Kan-2, validation of kanamycin resistance in knockout strains; lane 2: primer Kat-1 and Kat-2, validation of kanamycin resistance fragments and upstream and downstream homologous arm fragments in knockout strains; lane 3: primer Kat-1 and Kat-2, wild-type control）
Fig. 6 Construction and identification for katA-complemented strain HpΔkatA::katA A: Schematics of HpΔkatA::katA construction; B: Identification HpΔkatA::katA（lane 1: primer Chl-1 and Chl-2, validation of chloramphenicol resistance of the complementary strain; lane 2: primer Kat-3 and Kat-4, validation of chloramphenicol resistance fragments and upstream and downstream homologous arm fragments of the complementary strain）
Fig. 7 Growth curve of Hp（Helicobacter pylori）
Fig. 8 Hydrogen peroxide decomposing characteristics analysis of Hp
| 菌株Strain | 浓度Concentration/（mmol·L-1） | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| 0 | 3 | 6 | 10 | 20 | 40 | 60 | 80 | 100 | ||
| Hp_G272 | + | + | + | + | + | + | - | - | - | |
| HpΔkatA | + | + | + | - | - | - | - | - | - | |
| HpΔkatA::katA | + | + | + | + | + | + | - | - | - | |
Table 3 Analysis of tolerance of Hp to hydrogen peroxide
| 菌株Strain | 浓度Concentration/（mmol·L-1） | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| 0 | 3 | 6 | 10 | 20 | 40 | 60 | 80 | 100 | ||
| Hp_G272 | + | + | + | + | + | + | - | - | - | |
| HpΔkatA | + | + | + | - | - | - | - | - | - | |
| HpΔkatA::katA | + | + | + | + | + | + | - | - | - | |
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