Commentary
Ophuirid Ophiocomina Nigra HLA-E Gene Synthesis in PUC-GW-KAN Plasmid or HLA-E Echinodermata Gene Biosynthesis «De Novo» in E.Coli Sensu Lato Plasmid
Michel Leclerc*
Corresponding Author: Michel Leclerc, 556 rue Isabelle Romée, 45640 Sandillon, France.
Received: January 10, 2022; Revised: January 24, 2022; Accepted: January 27, 2022 Available Online: March 14, 2022
Citation: Leclerc M. (2022) Ophuirid Ophiocomina Nigra HLA-E Gene Synthesis in PUC-GW-KAN Plasmid or HLA-E Echinodermata Gene Biosynthesis «De Novo» in E.Coli Sensu Lato Plasmid. Int J Biopro Biotechnol Advance, 8(3): 457-458.
Copyrights: ©2022 Leclerc M. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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HLA-E (Class 1) is an MHC gene which has been isolated in 2020, in our laboratory. We show now its biosynthesis «de novo» in a PUC-GW-KAN plasmid. Such experiment was performed with the Ophiocomina nigra IGKappa gene one year ago.

INTRODUCTION

We have isolated recently MHC genes in Echinodermata [1] in 3 classes: the Ophuirids, the Crinoïds, the Asterids. At that time, we decided to synthetize one of these genes: the well-known HLA-E one in a PUC-GW-KAN plasmid (Yan Li gift).

METHODS

We operate according the following method [2]. It was resumed in 4 parts:
  1. Synthesis of oligonucleotides with overlapping segments in sense and antisense direction
  2. Assembly of the oligonucleotides into a double stranded DNA, using a poly chain assembly method (PCA).
  3. For larger constructs, the sequence is split into smaller, intermediate fragments, to facilitate synthesis. Once the intermediated fragments have been obtained with correct sequence, they are assembled into the full-length sequence.
  4. Cloning into the linearized vector by either recombination or ligation-based cloning, mostly performed within the same step as full-length sequence assembly.
Regarding the restriction site, which was used for cloning, construct was cloned into vector pUC-GW by using the unique EcoRV restriction site. Please find table below for the primers used for sequencing (Table 1).
RESULTS
  • Plasmid map
The construct appears below (Figure 1):
  • Recalling of Original sequence in 5’-3’:
TGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGCGGATCACGAGGTCAGGAGATCGAGACCATCCTGGCTAACACAGTGAAACCCCGTCTCTACTAAAAATACAAAAAATTAGCCGGGCGTGGTGGCGGGCGCCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAGGCGGAGCTTGCAGTGAGCCGAGATCGCGCCACTGCACTCCAGCCTGGGCGACAGAGCGAGACTCTGTCTCAAAAAAAAAAAAAAAAAAAAAAA
  • Synthetized sequence in 5’-3’:
TGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGCGGATCACGAGGTCAGGAGATCGAGACCATCCTGGCTAACACAGTGAAACCCCGTCTCTACTAAAAATACAAAAAATTAGCCGGGCGTGGTGGCGGGCGCCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAGGCGGAGCTTGCAGTGAGCCGAGATCGCGCCACTGCACTCCAGCCTGGGCGACAGAGCGAGACTCTGTCTCAAAAAAAAAAAAAAAAAAAAAAA
  • Blastn original sequence/ synthetized sequence
The Table 2 resumes mainly the identities and the e-value between these 2 precedent sequences. Chromatograms were also performed.



CONCLUSION
We conclude our experiment is valid when compared to Table 2. Furthermore, we assert, it is the first time such discovery:
  1. MHC Genes in Echinodermata (Invertebrates) were found
  2. biosynthesis of HLA-E Echinodermata gene in a PUC-GW-KAN plasmid was performed.
  1. Leclerc M (2020) Evidence of MHC Class I and Class II Genes in Echinodermata. Proteomics Bioinformatics 2(1): 59-61.
  2. Leclerc M (2021) Biosynthesis « De Novo » of the Ophuirid Ophiocomina Nigra Igkappa Gene. J Clin Class Immunol 1(1).
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