Difference between revisions of "Part:BBa K4195037"
(→Biology) |
|||
| (2 intermediate revisions by the same user not shown) | |||
| Line 2: | Line 2: | ||
__NOTOC__ | __NOTOC__ | ||
===Biology=== | ===Biology=== | ||
| − | + | ''Lv''APN1 | |
| + | |||
| + | ''Lv''APN1, a 999-residue membrane-bound protein from the aminopeptidase N family, was identified in the in-house transcriptome of ''Litopenaeus vannamei'' hemocytes. It is presumed that it contains a putative N-terminal transmembrane domain, the Cry-binding region (CBR), the peptidase-M1 domain, the GAMEN domain and the HEXXH(X)18E zinc-binding site motifs. It proves that the ''Lv''APN1 was involved in AHPND pathogenesis and acted as a VP<sub>AHPND</sub> toxin PirAB<sup>vp</sup> receptor by mediating the toxin penetration into hemocytes. ''Lv''APN1 knockdown reduced the mortality, histopathological signs of AHPND in the hepatopancreas (''1''). | ||
| − | |||
===Usage and design=== | ===Usage and design=== | ||
Considering that ''Lv''APN1 is a protein expressed by eukaryotic animals, we intended to use the X-33 wild-type strain of ''Pichia pastoris'' to express the receptor rather than ''E. coli''. In order to characterize ''Lv''APN1 on the cell surface of ''Pichia pastoris'', a His-tag (6×His) was added to the C-terminal of it. We used the common vector pPICZ B to express the ''Lv''APN1-his, of which the sequence was inserted between the ''Eco''R I and ''Xba'' I sites. The constructed plasmid were transformed into ''E. coli'' DH5α for plasmid propagation, then the positive transformants were selected by zeocin and confirmed by colony PCR and sequencing. | Considering that ''Lv''APN1 is a protein expressed by eukaryotic animals, we intended to use the X-33 wild-type strain of ''Pichia pastoris'' to express the receptor rather than ''E. coli''. In order to characterize ''Lv''APN1 on the cell surface of ''Pichia pastoris'', a His-tag (6×His) was added to the C-terminal of it. We used the common vector pPICZ B to express the ''Lv''APN1-his, of which the sequence was inserted between the ''Eco''R I and ''Xba'' I sites. The constructed plasmid were transformed into ''E. coli'' DH5α for plasmid propagation, then the positive transformants were selected by zeocin and confirmed by colony PCR and sequencing. | ||
===Characterization=== | ===Characterization=== | ||
| − | ==== | + | ====Identification==== |
After transforming the plasmid into ''E. coli'' DH5α, colony PCR was used to verify that the plasmid was correct. Target bands (3285 bp) can be observed at the position between 5000 bp and 3000 bp (Fig. 1). | After transforming the plasmid into ''E. coli'' DH5α, colony PCR was used to verify that the plasmid was correct. Target bands (3285 bp) can be observed at the position between 5000 bp and 3000 bp (Fig. 1). | ||
| Line 14: | Line 15: | ||
'''Fig. 1 DNA gel electrophoresis of the colony PCR products of LvAPN1-his_pPICZ B.''' | '''Fig. 1 DNA gel electrophoresis of the colony PCR products of LvAPN1-his_pPICZ B.''' | ||
| + | |||
===Reference=== | ===Reference=== | ||
1. W. Luangtrakul ''et al.'', Cytotoxicity of ''Vibrio parahaemolyticus'' AHPND toxin on shrimp hemocytes, a newly identified target tissue, involves binding of toxin to aminopeptidase N1 receptor. ''PLoS Pathog.'' '''17''', e1009463 (2021). | 1. W. Luangtrakul ''et al.'', Cytotoxicity of ''Vibrio parahaemolyticus'' AHPND toxin on shrimp hemocytes, a newly identified target tissue, involves binding of toxin to aminopeptidase N1 receptor. ''PLoS Pathog.'' '''17''', e1009463 (2021). | ||
Latest revision as of 12:49, 12 October 2022
Biology
LvAPN1
LvAPN1, a 999-residue membrane-bound protein from the aminopeptidase N family, was identified in the in-house transcriptome of Litopenaeus vannamei hemocytes. It is presumed that it contains a putative N-terminal transmembrane domain, the Cry-binding region (CBR), the peptidase-M1 domain, the GAMEN domain and the HEXXH(X)18E zinc-binding site motifs. It proves that the LvAPN1 was involved in AHPND pathogenesis and acted as a VPAHPND toxin PirABvp receptor by mediating the toxin penetration into hemocytes. LvAPN1 knockdown reduced the mortality, histopathological signs of AHPND in the hepatopancreas (1).
Usage and design
Considering that LvAPN1 is a protein expressed by eukaryotic animals, we intended to use the X-33 wild-type strain of Pichia pastoris to express the receptor rather than E. coli. In order to characterize LvAPN1 on the cell surface of Pichia pastoris, a His-tag (6×His) was added to the C-terminal of it. We used the common vector pPICZ B to express the LvAPN1-his, of which the sequence was inserted between the EcoR I and Xba I sites. The constructed plasmid were transformed into E. coli DH5α for plasmid propagation, then the positive transformants were selected by zeocin and confirmed by colony PCR and sequencing.
Characterization
Identification
After transforming the plasmid into E. coli DH5α, colony PCR was used to verify that the plasmid was correct. Target bands (3285 bp) can be observed at the position between 5000 bp and 3000 bp (Fig. 1).
Fig. 1 DNA gel electrophoresis of the colony PCR products of LvAPN1-his_pPICZ B.
Reference
1. W. Luangtrakul et al., Cytotoxicity of Vibrio parahaemolyticus AHPND toxin on shrimp hemocytes, a newly identified target tissue, involves binding of toxin to aminopeptidase N1 receptor. PLoS Pathog. 17, e1009463 (2021).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2741
Illegal XhoI site found at 574 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]