[Schistosoma japonicum ferritin: cloning, nucleotide sequencing, expression, and purification].
Our previous work showed that immunization of mice with Schistosoma japonicum (Sj) immature eggs induced significant immunity against fecundity and embryonation of the parasite. The Sj adult cDNA library was screened by sera from rabbits against Sj immature egg antigen (RASjIEA). The genes encoding molecules which may induce immunity against fecundity/embryonation were chosen for further cloning and expression. First of all, RASjIEA was absorbed with E. coli lysate to remove cross reactive antibodies. The cDNA library was then immunoscreened using the routine method. The resulted positive plaques were rescreened till individual clones were confirmed. Phagemids were obtained using in vivo excision. The positive clones were amplified using PCR. The sizes of the genes were determined by agarose gel electrophoresis. After DNA sequencing of the genes cloned, Gene bank was searched and six different genes were identified from a total of 102 positive clones. One of six identified genes, Sj ferritin (SjFer) was chosen to subclone into pGMC vector. According to DNA sequences of Sj Fer and MCS (multiple cloning site) of the vector, forward primer (Fer/GMC1) and reverse primer (Fer/GMC2) were designed and used to amplify Sj Fer by PCR. The Sj Fer cDNA and expression vector pGMC were digested with BamHI and XhoI. The digested cDNA and pGMC were ligased by T4 DNA ligase to construct a recombinant which was then used to transform E. coli strain ER2566. The fusion protein GMCSF-Sj Ferritin was expressed in insoluble form, the inclusion body. Pellets were harvested and resolved in Tris-HCl buffer containing 8M urea. GMCSF-Sj Ferritin was purified by affinity chromatography using Ni-NTA resin. The molecular weight was determined by SDS-PAGE. This study first reports the gene encoding S. japonicum ferritin as a new candidate for schistosome vaccine.
Yi
X
X
Department of Parasitology, Hunan Medical University, Changsha.
Zeng
X
X
Zhou
J
J
Cai
C
C
Shu
X
X
Yang
W
W
McReynolds
L
L
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018509:DNA, Helminth / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005293:Ferritins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D015870:Gene Expression; D017238:Genes, Helminth; D051379:Mice; D008969:Molecular Sequence Data; D011817:Rabbits; D011993:Recombinant Fusion Proteins; D012549:Schistosoma japonicum / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA
D018509:DNA, Helminth; D011993:Recombinant Fusion Proteins; D005293:Ferritins