[Expression of single chain fragment variable P1D3 antibody against shrimp white spot syndrome virus in Pichia pastoris].
White spot syndrome virus (WSSV) is a major pathogen in aquaculture penaeid shrimp, which caused catastrophic economic losses in the worldwide. No adequate treatments against WSSV are available. In order to study infection mechanism of WSSV, a phage display scFv cDNA library against WSSV was constructed and a neutralizing antibody of scFv P1D3 was selected in our lab previously. In this study, scFv P1D3 was expressed successfully in yeast. Firstly, the original expression vector of P1D3, M13 phagmid, was used as a template to design primers with restriction sites of SnaB I and EcoR I . Then the gene of P1D3 was amplified by PCR. After digested by SnaB I and EcoR I , the fragment of scFv P1D3 with E-tag was inserted into yeast and E. coli shuttle plasmid pPIC9k. The recombinant plasmid pPIC9k-scFv P1D3-Etag was linearized with Bgl II and then transformed into Pichia pastoris GS115 by electroporation. Positive clones were selected and verified by PCR and DNA sequencing. The scFv PID3 was induced to express in yeast by methanol. The results of ELISA demonstrate that scFv P1D3 expressed in yeast still has high specificity to bind on WSSV and the binding activity is higher than that expressed in E. coli TG1. After several optimizing experiments, the results show that the expression amount of scFv P1D3 can reach to 302 mg/L in yeast culture supernatant. This experiment has offered a new source of antibody for the researches on passive immunology for shrimp.
Yang
Yi
Y
Institution of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.
Zhang
Min
M
Yuan
Li
L
Zhang
Xiao-Hua
XH
Dai
He-Ping
HP
D000818:Animals; D000918:Antibody Specificity; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression / Q000187:drug effects; D000432:Methanol / Q000494:pharmacology; D033561:Penaeidae / Q000821:virology*; D010843:Pichia / Q000187:drug effects / Q000235:genetics*; D057127:Single-Chain Antibodies / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D013696:Temperature; D046848:White spot syndrome virus 1 / Q000276:immunology*
D057127:Single-Chain Antibodies; D000432:Methanol