This study was purposed to construct a fusion DNA vaccine containing WT1 multi-epitope and stimulating epitope of mycobacterium tuberculosis heat shock protein 70 and to detect its expression and immunogenicity. On the basis of published data, a multi-epitope gene (Multi-WT1) containing three HLA *0201-restricted CTL epitopes: one HLA*2402-restricted CTL epitope, two Th epitopes and one universal Th Pan-DR epitope (PADRE) was constructed. DNA-coding sequence was modified by Computer-Aided Design (CAD) to optimize proteasome-mediated epitope processing through the introduction of different amino acid spacer sequences. The synthetic nucleotide sequence was then inserted into an eukaryotic vector to construct the plasmid pcDNA3.1-WT1.For enhancing CTL activity, HSP70 fragment including stimulatory domain P407-426 was amplified by PCR from mycobacterial HSP70 gene and cloned into pcDNA3.1(+). Then Multi-WT1 was fused to the N-terminal of pcDNA3.1-mHSP70(407-426) to make the multi-epitope fusion gene vaccine pcDNA3.1-WT1-mHSP70(407-426). HEK-293T cells were transfected with this vaccine and the expressed product was identified by RT-PCR. Enzyme-linked immunospot assay (ELISPOT) was used to evaluate the immunological responses elicited by vaccine. The results showed that the most of WT1 epitopes could be correctly cleaved which was confirmed by software Net Chop 3.1 and PAPROCIanalysis. RT-PCR showed correct expression of target gene in HEK293T cells and ELISPOT showed specific T-cell responses. It is concluded that the eukaryotic expression vector PcDNA3.1-WT1-mHSP70(407-426) fusion gene has been successfully constructed and the immunity response is also elicited, which is a good candidate for further research of DNA vaccine.
Bacterial Proteins ; genetics ; immunology ; Epitopes ; genetics ; immunology ; Genetic Vectors ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; Humans ; Immunodominant Epitopes ; Vaccines, DNA ; genetics ; immunology ; WT1 Proteins ; genetics ; immunology

ObjectiveTo establish the specific chromatogram and thin layer chromatography（TLC） identification method of Kaixinsan（KXS） samples， in order to clarify the key quality attributes and provide reference for the quality evaluation of KXS. MethodHigh performance liquid chromatography（HPLC） specific chromatogram of KXS was developed with YMC Hydrosphere C₁₈ column（4.6 mm×250 mm， 5 μm）， the mobile phase was acetonitrile（A）-0.2% formic acid aqueous solution（B） for gradient elution（0-15 min， 2%-20%A； 15-25 min， 20%-25%A； 25-30 min， 25%-30%A； 30-45 min， 30%-31%A； 45-50 min， 31%-44%A； 50-65 min， 44%-45%A； 65-73 min， 45%-75%A； 73-95 min， 75%-100%A； 95-105 min， 100%A； 105-105.1 min， 100%-2%A； 105.1-120 min， 2%A）， the detection wavelength was 320 nm. Ultra high performance liquid chromatography-linear ion trap-electrostatic field orbitrap mass spectrometry（UHPLC-LTQ-Orbitrap MS） was used to identify the chemical components of KXS with electrospray ionization（ESI）， negative ion mode and scanning range of m/z 50-2 000. TLC identification methods for Poria and Ginseng Radix et Rhizoma in KXS were established. ResultThere were 11 common peaks in the specific chromatogram of KXS， attributed to Polygalae Radix， Poria and Acori Tatarinowii Rhizoma. Taking peak 9（α-asarone） as the reference peak， the relative standard deviations of the retention times of 15 batches of KXS samples were<0.2%. A total of 34 compounds were identified by UHPLC-LTQ-Orbitrap MS， including terpenoids， phenylpropanoids， oligosaccharides and ketones. The established TLC had good separation and was rapid， reliable， simple， feasible， suitable for the identification of Poria and Ginseng Radix et Rhizoma in KXS. ConclusionThe specific chromatogram and TLC of KXS are stable and reproducible. The material basis of KXS is basically clarified by MS， which can provide a reference for the development and quality control of KXS.