Sec at 94☌, 30 sec at 55☌ and 90 sec at 72☌ for 30 cycles, HSPN, HSPC and HSP were incubated for 3 min at 94☌, followed by 30 PSCAĪmplification was performed for 3 min at 94☌, immediately followedīy 30 sec at 94☌, 30 sec at 55☌ and 30 sec at 72☌ for 30 cycles. PET21-PSCA-HSPN, pET21-PSCA-HSPC and pET21-PSCA-HSP. Various structural domains of HSP70 were cloned into theĮcoRI and XhoI sites of pET21-PSCA to generate GAA TTC ATG GCC AAA GCC GCG GCG-3′ and 5′-CCG CTC GAG ATC TAC CTCĬTC AAT GGT-3′, respectively. The primers 5′-CCG GAA TTC ATG GCC AAA GCC GCG GCG-3′ and 5′-CCGĬTC GAG TCG CTT GTT CTG GCT GAT-3′, 5′-CCG GAA TTC CTG AAC AAG AGCĪTC AAC-3′ and 5′-CCG CTC GAG ATC TAC CTC CTC AAT GGT-3′, 5′-CCG Length genes of HSP70 were amplified from the pMD-HSP70 vector with The amino-terminus, carboxyl-terminus and overall NdeI and EcoRI sites of pET21a(+) to generate

With the primers 5′-CCG CAT ATG TGC TAT AGC TGC AAA GCC-3′ andĥ′-CCG GAA TTC CAG GGC ATG GGC CCC GCT-3′ and cloned into the The human PSCA gene was amplified from the pMD-PSCA vector Construction of expression plasmidsĪll constructions were cloned into the pET21a(+) The study was approvedīy the ethics committee of the Academy of Military Medical Sciences Used in this study were of analytical grade. Anti-PSCA polyclonalĪntibody and fluorescein isothiocyanate (FITC)-conjugated goatĪnti-rabbit IgG antibody were purchased from Santa Cruzīiotechnology, Inc. The restriction enzyme were purchased from Takara BiotechnologyĬo., Ltd. coli) DH5αĪnd BL21(DE3), pMD18-T, LA Taq, T4 DNA ligase, DNA marker 2000 and Stored in the Beijing Institute of Biotechnology, Academy of The plasmids pET21a(+), pMD-HSP70 and pMD-PSCA were Recombinant proteins with a purity >95%, which lays theįoundation for the development of a vaccine for prostateĬancer. Recombinant plasmids based on PSCA and HSP70 and obtained soluble In the present study, we successfully constructed Involved in the process of antigen presentation and has potentialĪs a chaperone for specific antigens in vaccines. HSP70-peptide complexes elicit specific antitumor responses Previous studies have demonstrated that vaccination with It is also involved in theįunctions of the mitochondria, endoplasmic reticulum and nucleus. Heat shock protein-70 (HSP70) is a major molecularĬhaperone, which assists in transport, folding and assembly of Target for prostate cancer immunotherapy. Responses and inhibits the growth of PSCA-expressing tumors in mice Vaccination based on PSCA enhances the cellular and humoral immune There is a direct correlation between the expression level of PSCAĪnd tumor stage, grade and bone metastasis ( 2). In 85% of prostate cancer specimens with high tissue specificity. Surface glycoprotein and has a 30% homology with stem cell Prostate stem cellĪntigen (PSCA), first described by Reiter et al, is a Therapeutic strategy for cancer treatment. Studies on novel anticancer strategies have introduced prostateĬancer immunotherapy, which represents a highly attractive Prostate cancer is the most common non-cutaneousĬancer with a high mortality rate in American males. The purity of the recombinant proteins PSCA‑HSPC and PSCA-HSP reached >95% following purification with the nickel-nitrilotriacetic acid (Ni-NTA) resin, Phenyl-Sepharose Fast Flow and Superdex 75, which lays a foundation for the development of vaccines for prostate cancer. The PSCA-HSPC and PSCA-HSP expressed in E. coli existed in soluble form, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results revealed that recombinant plasmids were successfully constructed. Western blotting was performed to confirm the expression of the recombinant proteins. After being expressed in Escherichia coli (E. coli) by isopropyl β-D-1-thiogalactopyranoside (IPTG) induction, recombinant fusion proteins were purified. Then, the PSCA was cloned into the prokaryotic expression vector pET21a(+) with the amino-terminus, carboxyl-terminus and overall length of HSP70, by enzyme digestion to construct the recombinant plasmids pET21-PSCA-HSPN, pET21-PSCA-HSPC and pET21-PSCA-HSP, respectively. The PSCA gene and various structural domains of HSP70 were amplified by polymerase chain reaction (PCR) with the respective primers. The aim of this study was to express and purify recombinant proteins based on human prostate stem cell antigen (PSCA) and heat shock protein-70 (HSP70).

Protein ladder on western blot dnak