According to the expected epitopes and secondary structure and functional fragments [24], we synthesized nine peptides. experiments. This study was carried out in strict accordance with the recommendations in the of the Ministry of Technology and Technology of Peoples Republic of China. Protocol BIME 2014-16 was authorized by the Institutional Animal Care and Use Committees of Beijing Institute of Microbiology and Epidemiology. 2.2. Evaluation of Adjuvanticity of TFPR1 for the HLCL-61 Peptide Antigen HIV-1 CBD Adjuvanticity of TFPR1 was evaluated in female 6- to 8-week-old BALB/c mice using the synthetic peptide antigen HIV-1 CBD (SLEVIWNNMTWMEWEREIDN), which was derived from the caveolin-binding website of HIV-1 gp41 [28,29]. Mice (= 6 per group) were immunized intramuscularly with aqueous combination comprising 20 g of HIV-1 CBD and 10 g of recombinant TFPR1, or HIV-1 CBD adsorbed to alum (InvivoGen, volume percentage of OVA to alum is definitely 1:9), HIV-1 CBD only, or endotoxin-free PBS (GIBCO, Grand Island, NY, USA), and boosted once three weeks later on. Serum was collected before immunization and on the second week after the final immunization to detect specific anti-HIV-1 CBD antibodies IgG, IgG1 and IgG2a by ELISA, HIV-1 CBD at a final concentration of 1 1 g/mL was used as the capture antigen. The protocol is similar to that explained previously [30]. The antibody titer was defined as the reciprocal of the largest dilution of serum which OD450 value was greater than average value of the bad control (before immunization) plus two-fold of the standard deviation HLCL-61 (SD). 2.3. Analysis and Recognition of B Cell Epitopes on TFPR1 The B cell epitopes on TFPR1 were expected using IEDB on-line software (http://tools.iedb.org/main/bcell/), and the structural and functional motifs of TFPR1 were analyzed from the tool secondary structure consensus prediction of NPS (http://npsa-prabi.ibcp.fr) and according to the research [24]; then, the peptides which were the expected B cell epitope and/or key motifs of TFPR1 were synthesized (Genscript, Nanjing, China), and used as covering antigens in ELISA for further epitope recognition; serum was collected from your TFPR1- or TFPR1/Alum-immunized BALB/c mice. The immunization was performed as the following: 6- to 8-week-old female BALB/c mice were immunized intramuscularly with 10 g of TFPR1 only or TFPR1 plus alum, and endotoxin-free PBS only was used as bad control. Serum specific antibody IgG to each peptide were measured by ELISA, the 96-well plate was precoated with each synthetic peptide (2 g/mL) separately, and TFPR1 was used as positive control, the serum was diluted 1/20 and added into each well. Data was offered as the OD450 value, the epitope was defined as positive relating to one-way ANOVA analysis compared with bad control. 2.4. Manifestation, Purification, and Structure Prediction of Different Truncated Fragments of TFPR1 As explained previously [26,27,28,29,30], the truncated fragments of TFPR1 were designed and put into the pQE-30 vector (comprising 6 His-tag coding sequence), and indicated in E.coli. Because the recombinant proteins were expressed in the form of inclusion bodies, after the proteins were purified by Ni-NTA chromatography and dissolved in 8 M urea, the soluble denatured proteins were refolded by gradient dialysis in 6 M, 4 M and 2 M urea in tris-glycine buffer, followed by Laemmli buffer and PBS. The manifestation of HLCL-61 recombinant truncated proteins was analyzed by SDS-PAGE and confirmed by Western blot with mouse sera comprising anti-TFPR1 antibody [30]. Finally, the purified proteins were treated having a lipopolysaccharide (LPS)-eliminating gel (Detoxigel?, Pierce Biotechnology, Rockford, IL, USA) to remove the contaminating LPS. The endotoxin content of the purified proteins was then tested using the Limulus amebocyte lysate (LAL) assay, and the proteins MLNR which tested bad were utilized for the following experiments. The conformational constructions of TFPR1 and its truncated fragments were expected by online software SWISS-MODEL (http://swissmodel.expasy.org/), difference between the HLCL-61 full-length protein TFPR1 and its truncated fragments was compared. The secondary structure of each protein was analyzed using Circular Dichroism [31,32] (Chirascan, Applied Photophysics Ltd, England), and analyzed using CDNN software (Gerald B?hm, Magdeburg, England). 2.5. Adjuvanticity Evaluation of Each Truncated Fragment of TFPR1 Using Mouse Bone MarrowCDerived Dendritic Cells in Vitro Bone marrow cells were collected from 4- to 5-week-old healthy male BALB/c mice under aseptic conditions, and were cultured at a denseness of 2.5 million per mL in 96-well plates with complete RPMI 1640 (GIBCO, Grand Island, NY, USA) containing rmGM-CSF (final concentration, 10 ng/mL) and rmIL-4 (final.
