Prokaryotic expression of a recombinant protein of adeno-associated virus capsid conserved regions and preparation of its polyclonal antibody / 南方医科大学学报

OBJECTIVE@#To express and purify the antigenic peptide of adeno-associated virus (AAV) capsid conserved regions in prokaryotic cells and prepare its rabbit polyclonal antibody.@*METHODS@#The DNA sequence encoding the conserved regions of AAV capsid protein was synthesized and cloned into the vector pET30a to obtain the plasmid pET30a-AAV-CR for prokaryotic expression and purification of the conserved peptides. Coomassie blue staining and Western blotting were used to identify the AAV conserved peptides. Japanese big ear white rabbits were immunized with AAV conserved region protein to prepare polyclonal antibody, with the rabbits injected with PBS as the control group. The antibody titer was determined with ELISA, and the performance of the antibody for recognizing capsid protein sequences of AAV1-AAV10 was assessed with Western blotting and immunofluorescence assay.@*RESULTS@#The plasmid pET30a-AAV-CR was successfully constructed, and a recombinant protein with a relative molecular mass of 17000 was obtained. The purified protein induced the production of antibodies against the conserved regions of AAV capsid in rabbits, and the titer of the purified antibodies reached 1:320 000. The antibodies were capable of recognizing a wide range of capsid protein sequences of AAV1-AAV10.@*CONCLUSION@#We successfully obtained the polyclonal antibodies against AAV capsid conserved region protein from rabbits, which facilitate future studies of AAV vector development and the biological functions of AAV.

Predominant antigenic epitopes on Hap adhesin of nontypeable Haemophilus influenzae and their immunogenicity / 中华微生物学和免疫学杂志

Objective To investigate the distribution and sequence conservation of Hap adhensin encoding gene (hap) in clinical isolates of nontypeable Haemophilus influenzae (NTHi), to screen out and identify the predominant T-and B-cell (T-B) combined antigenic epitopes on Hap protein and to analyze their immunogenicity.Methods Sequence conservation of hap genes in NTHi strains and T-B combined antigenic epitopes were predicted using bioinformatic softwares.PCR was used to amplify the 156 bp segment at 5′-end and the 855 bp segment at 3′-end of hap gene (hap-5′-156 and hap-3′-855) and the amplified products were sequenced.Phage display systems of seven T-B combined antigenic epitopes located on the 55 aa segment at N-terminal and the 285 aa segment at C-terminal of Hap protein (Hap-N52 and Hap-C285) were constructed.Western blot assay and ELISA were performed to detect the antigenicity and immunoreactivity of different T-B combined epitopes displayed by recombinant phage PⅢ protein (rPⅢ).Results Hap protein encoded by the hap gene in NTHi was located on membrane surface.Sequences of the 156 bp segment at 5′-end and the 855 bp segment at 3′-end of hap genes extracted from different NTHi strains were relatively conservative, but many mutations were found in sequences at the middle regions of these hap genes.All of the 56 NTHi strains carried hap-5′-156 and hap-3′-855 segments and shared 92.3％-100％ identities in nucleotide and amino acid sequences of these segements.Hap-N5-24 in the Hap-N52 segment as well as Hap-C4-27, Hap-C28-47, Hap-C114-129, Hap-C150-173, Hap-C200-227 and Hap-C241-267 in the Hap-C285 segment was predicted as the T-B combined antigenic epitope with a higher score and less mutations.Results of Western blot assay and ELISA confirmed that the rPⅢ-displayed Hap-C4-27 and Hap-C150-173 epitopes presented clear hybridization bands with NTHi antisera, and 96.9％ (63/65) and 92.3％ (60/65) of serum samples from children with NTHi infection were positive for antibodies against Hap-C4-27 and Hap-C150-173 epitopes, respectively.Conclusion The gene of hap is widely distributed in clinical isolates of NTHi.Moreover, sequences of the 156 pb segment at 5′-end and the 855 bp segment at 3′-end of hap gene are conservative.Hap-C4-27 and Hap-C150-173 are the predominant T-B combined antigenic epitopes on Hap protein, suggesting that they can be used as epitope candidates for developing multiple antigenic peptide vaccines against NTHi.

Predominant T-B-combined epitopes and their immunogenicity of P6 outer membrane protein of nontypeable Haemophilus influenzae / 中国人兽共患病学报

The aim of this study is to investigate the distribution and sequence conservation of P6 outer membrane protein (OMP6)-encoding gene of nontypeable Haemophilus influenzae isolates as well as screen and identify the predominant T-and B-cell (T-B)-combined antigenic epitopes in OMP6 sequences and their immunogenicity.The entire omp6 genes of NTHi isolates were amplified by PCR and the amplification products were sequenced after T-A cloning.By using bioinformatic softwares,the sequence conservation and membrane location of OMP6 were analyzed as well as the T-B-combined antigenic epitopes in OMP6 were predicted.The immunogenicity and immunoreactivity of T-B-combined antigenic epitope peptides displayed by recombinant phage PⅢ proteins (rPⅢ) were determined by Western Blot assay and ELISA.The PCR showed that all the 35 NTHi isolates tested were detectable for omp6 gene.The identities of nucleotide and amino acid sequences of omp6 genes from 28 strains in the NTHi isolates were 98.3％-100％ and 99.3 ％-100％,respectively.OMP6 of NTHi was predicted as an outer membrane superficial protein that contains OMP6-2-25,OMP6-61-86 and OMP6-98-126 predicted T-B-combined antigenic epitopes.The immunoblotting assay and ELISA confirmed that OMP6-2-25 presented stronger hybridization band with NTHi antisera while 96.9％ (59/62),69.4％ (43/62) and 74.2％ (46/62) of serum samples from NTHi-infected children were positive for OMP6-2-25,OMP6-61-86 and OMP6-98-126 T-B-combined antigenic epitope peptides,respectively.All the results lead to a conclusion thatomp6 is an extensive distribution and sequence conserved gene of NTHi,and OMP6-2-25 is the predominant T-B-combined antigenic epitopes which can be used as the candidates for developing multiple antigenic peptide vaccine against NTHi.

A novel M2e-multiple antigenic peptide providing heterologous protection in mice

Swine influenza viruses (SwIVs) cause considerable morbidity and mortality in domestic pigs, resulting in a significant economic burden. Moreover, pigs have been considered to be a possible mixing vessel in which novel strains loom. Here, we developed and evaluated a novel M2e-multiple antigenic peptide (M2e-MAP) as a supplemental antigen for inactivated H3N2 vaccine to provide cross-protection against two main subtypes of SwIVs, H1N1 and H3N2. The novel tetra-branched MAP was constructed by fusing four copies of M2e to one copy of foreign T helper cell epitopes. A high-yield reassortant H3N2 virus was generated by plasmid based reverse genetics. The efficacy of the novel H3N2 inactivated vaccines with or without M2e-MAP supplementation was evaluated in a mouse model. M2e-MAP conjugated vaccine induced strong antibody responses in mice. Complete protection against the heterologous swine H1N1 virus was observed in mice vaccinated with M2e-MAP combined vaccine. Moreover, this novel peptide confers protection against lethal challenge of A/Puerto Rico/8/34 (H1N1). Taken together, our results suggest the combined immunization of reassortant inactivated H3N2 vaccine and the novel M2e-MAP provided cross-protection against swine and human viruses and may serve as a promising approach for influenza vaccine development.

Preparation of high specific antibody against human C1q/TNF-related protein-1 / 国际药学研究杂志

Objective: To prepare specific antibody against human C1q/TNF-related protein-1 (hCTRP1). Methods: A C-terminal antigenic peptide of hCTRP1 was synthesized and injected into the Newzealand rabbits. The antibody was purified by affinity chromatography column. Results: High titer antisera against the hCTRP1 was prepared and the antiserum's titration was over 1: 64 000 by indirect ELISA. The specificity of the purified antibody by affinity chromatography was identified by Western blot and ELISA. Conclusion: The high titer antiserum is prepared by injection for rabbits and the specificity of the antibody purified from the antiserum is determined.

Preparation and clinical application of a novel monoclonal antibody against Mycobacterium tuberculosis culture filtrate protein 10 / 中华临床感染病杂志

Objective To prepare a novel monoclonal antibody (mAb) that specifically against Mycobacterium tuberculosis culture filtrate protein 10 (CFP-10).Methods The BALB/c mice were immunized by a peptide with 14 amino acids (aa residues 53 to 66) of CFP-10,and then the splenocytes of mice were fused with myeloma cell line SP2/0.The resultant fused cells were subjected to screening culture,enzyme linked immunosorbent assay (ELISA) assay and subcloning by limited dilution to establish hybridoma cell lines of stable secreting anti-the peptide of CFP-10 antibody.The antibody was purified,and its isotypes were analyzed.Then,the antibody was further evaluated by Western blotting,immunoprecipitation and ELISA in 38 culture supernatant samples of Mycobacterium tuberculosis,20 culture supernatant samples of non-Mycobacterium tuberculosis,32 samples of tuberculous pleural effusion,24 samples of non-tuberculous pleural effusion,and 20 serum samples from healthy controls.Results The isotype of the mAb against the specific peptide of CFP-10 was an IgG1 with κ chain,and it was applicable for Western blotting and immunoprecipitation analysis.ELISA quantitative test showed that the sensitivity and specificity for diagnosis of Mycobacterium tuberculosis were 78.6％ (55/70) and 92.2％ (59/64),respectively.Conclusion The mAb generated against the specific peptide of CFP-10 is high in sensitivity and specificity,and it might be used in the early diagnosis of tuberculosis.

Synthesis of multiple antigenic peptide vaccine based on predominant epitopes of Helicobacter pylori UreB protein and immunoprotection of the vaccine / 中华微生物学和免疫学杂志

ObjectiveTo generate a prokaryotic expression system of series predominant epitopes (UreB322 and UreB527) of Helicobacter pylori UreB protein,and to synthesize a multiple antigenic peptide (MAP) vaccine by linking both the two epitopes with a peptide carrier (Poly-Asp-Lys),and to determine the immunogenicity and immunoprotection of the MAP vaccine.MethodsLinking primer PCR was performed to generate an enterokinase(EK) site-containing series UreB322 and UreB527 epitope encoding gene for construction of its prokaryotic expression system.The expressed target recombinant fusion protein 8 ×[rEK-UreB322-EK-UreB527-EK] was hydrolyzed with EK and then rUreB322-EK and rUreB527-EK epitope peptides were extracted using a Sephadex G-25 column.rUreB322-EK,rUreB527-EK and Poly-Asp-Lys were linked using carbodiimide method to produce a MAP vaccine (MAP-rUreB322/B527).The antigenicity and immunoreactivity of each of the two epitope peptides and MAP-rUreB322/527 were determined by ELISA and Western blot assay.An animal H.pylori strain SS1-infected model in BALB/c mice was used to detect the immunoprotection of MAP-rUreB322/527.ResultsAn octuple-repeated series UreB322-UreB527 encoding gene and its prokaryotic expression system were obtained.The yield of target fusion protein 8×[rEKUreB322-EK-rUreB527-EK] was as high as 48％ of the total bacterial proteins.EK hydrolyzed the target fusion protein completely into rUreB322-EK and rUreB527-EK peptides.The linking ratio of rUreB322-EK,rUreB527-EK and Poly-Asp-Lys was as high as 92.5％.The antibody against whole cell of H.pylori and rUreB-IgG could recognize and combine with the rUreB322-EK,rUreB527-EK or MAP-rUreB322/527.The specific serum antibody level in MAP-rUreB322/527-immunized mice was significantly higher than that in rUreB-immunized mice (P＜0.05).The immunoprotective rates(83.3％ and 91.7％ ) by immunization with50 or 100 μg MAP-rUreB322/527 in the H.pyloristrain SSI-infected mice were significantly higher that those(d1.7％ and 50.0％ ) by immunization with equal rUreB(P＜0.05).ConclusionAn gene composed for encoding a repeated series predominant epitopes of H.pylori UreB protein and its prokaryotic expression system are successfully generated in this study.MAP-rUreB322/527,the multiple antigenic peptide vaccine based on the two predominant epitopes of UreB,can noticeably increase the immunoprotection in H.py/or/infected mice.

Altered tumor antigen peptide vaccines and their application in oncotherapy / 国际肿瘤学杂志

Tumor antigenic peptide vaccines,which have been adapted with altered peptide ligand (APL) and adjuvant to improve cytotoxic T lymphocyte response,include vaccines of different originations. These vaccines are orginated from malignant melanoma differentiation, HER2, NY- ESO-1, carcinoembryonic antigen and etc. Recently,great advances have been made in the related alteration methods,peptide designing principles and cytotoxic T lymphocyte response improvement mechanisms of these peptide vaccines. Furthermore,the vac- cines are showing satisfying pedormances in clinical treatments.

Construction of multiple antigenic peptide and the immunity analysis / 中华微生物学和免疫学杂志

Objective To construct the multiple antigenic peptide (MAP) gene and E. coli ex-pression system, based on the out membrane protein OmpL1, LipL21 and LipL32 from Leptospira interro-gans, and better understanding of the immunological activity of the recombinant protein. Methods Using MI3KE display and Western blot, the advantage epitopes of OmpL1, LipL21 and LipL32 were identified and used to synthesize a new gene, then its prokaryotic expression system was constructed. The expression of re-combinant protein was determined by SDS-PAGE. The immunity activity of the recombinant protein was iden-tified by Western blot and ELISA. Results The synthetic gene was effectively expressed in E. coli and mainly presented in soluble form. The expression protein could react with the antileptospirosis antibodies in rabbit and human sera, which contained different serogroups. Conclusion The recombinant MAP gene of leptospires was successfully constructedand and expressed in E. coli. The recombinant protein had a good immune activity, and could cross-reacted with antileptospirosis antibodies from different serogroups.

Antitumor Responses Induced by Recombinant Vaccinia Viruses Expressingp53 Antigenic Peptide and B7 / 中国肿瘤生物治疗杂志

Objective: To explore antitumor responses induced by recombinant vaccinia viruses expressing a p53 antigenic peptide (rVV-p53M) and enhanced effect of recombinant vaccinia viruses expressing costimulatory molecule B7 (rVVB7). Methods: A 135 Cys to Tyr point mutation p53-transduced P815 mastocytoma (P815-mp53) was used as an experimental tumor and the p53 protein as the model of tumor associated antigen． rVV-p53M and rVV-B7 were used as vaccine to test their induction of CTL and antitumor immunity. Results: Immunization of BABL/c mice with rVV-p53M could elicit specific CTLs, which could specifically lyse P815-mp53 cells. Immunization of mice with rVV-p53M could survive a part of mice challenged with 1×106 P815-mp53. Treatment with rVV-p53M could significantly prolong the survival oftumor-bearing mice. Admixture at 1:1 ratio of rVV-p53 M and rVV-B7 could enhance antitumor responses induced by rVV-p53M. Conclusion: Immunization with recombinant vaceinia virus expressing antigenic peptide is a useful alternative to peptide-based vaccine. Costimulatory molecule B7 can enhance antigenic peptide to induce antitumor responses.

Induction of specific cytotoxic T lymphocytes using hepatoma antigenic peptide mixed with HSP70 in vitro / 中国免疫学杂志

Objective:To explore the possibility of inducing cell mediated immune response with HSP70 antigenic peptide complex in vitro.Methods:HSP70 peptide complex was reconstituted in vitro.Granulocyte/macrophage colony stimulating factors and interleukin 4 were used to cultivate DC from peripheral blood of HLA A2 positive healthy donors.HSP70,HSP70 peptide complex or peptide was used to activate the DC individually,which will initiate homogenize T lymphocyte to form cytotoxic T lymphocyte(CTL).The cytotxicity of the CTL was detected by MTT assay.Results:It was found that peptide specific CD8 + CTL responses were readily elicited by HSP70 peptide complex or peptide.The CTL response primed by HSP70 peptide complex was more potene than peptide alone.Conclusion:The results suggest that HSP70 peptide complex is immunogenic and HSP70 can lead to great efficient CTL response,antigenic peptides and HSP70 complex may be used as peptide vaccines for cancer immunotherapy.

The study of human melanoma-specific antigen peptides by HLA-A2 restricted tumor-infiltrating lymphocytes / 中国免疫学杂志

Objective:To explore the characteristics of human melanoma-specific antigen peptides by HLA-A2 restricted tumor-infiltrating lymphocytes.Methods:The HLA-A2 protein and polypeptides molecules were purified from the three tumor cell lines(624-Mel, Chap-Mel and JY) by immunoaffinity chromatography, after the peptides bound to HLA-A2 protein solution were acidified with acetic acid and boiled by high temperature, and centrifuged through an Ultra-CL filter, then the peptides extracts were fractionated by revered phase high pressure liquid chromatography(RP-HPLC). Individual fractions were assessed for their ability to reconstitute melanoma-specific epitopes by adding to the HLA-A2 Ag-procceing mutant cell, T2. The biological feature of one of three active peptides from RT-HPLC samples was performed by mass spectrometric analysis. The synthetic peptides identical to active peptide sequences were determined in the reconstitute test.Results:Three prominent peaks(P19, P25 and P31) of the fraction from 624-Mel were observed in the reconstitute test, TIL killing rate was 67% for (P31) peptide fraction. The mass spectrometric analysis of one of active peptides (P31) showed that at mass-to-charge ratio(m/z) 948 has been usually nine residues. The sequence is H+ Ala Lue Trp Lue Phe Phe Gly Val Lue OH-. The peptide synthesized comprising epitopes were verified.Conclusion:These results showed the peptides derived from active fractions were related to human melanoma-specific tumor antigen peptides recognized by HLA-A2-restriced TIL. These peptides could develop novel peptide-based an anti-tumor vaccine for immunotherapy of CTL.

Antitumor Responses Induced by Recombinant Vaccinia Viruses Expressing p53 Antigenic Peptide and B7 / 中国肿瘤生物治疗杂志

Objective: To explore antitumor responses induced by recombinant vaccinia viruses expressing a p53 antigenic peptide (rVV p53 M) and enhanced effect of recombinant vaccinia viruses expressing costimulatory molecule B7 (rVV B7). Methods: A 135 Cys to Tyr point mutation p53 transduced P815 mastocytoma (P815 mp53) was used as an experimental tumor and the p53 protein as the model of tumor associated antigen. rVV p53 M and rVV B7 were used as vaccine to test their induction of CTL and antitumor immunity. Results: Immunization of BABL/c mice with rVV p53 M could elicit specific CTLs, which could specifically lyse P815 mp53 cells. Immunization of mice with rVV p53 M could survive a part of mice challenged with 1?10 6 P815 mp53. Treatment with rVV p53 M could significantly prolong the survival of tumor bearing mice. Admixture at 1∶1 ratio of rVV p53 M and rVV B7 could enhance antitumor responses induced by rVV p53 M. Conclusion: Immunization with recombinant vaccinia virus expressing antigenic peptide is a useful alternative to peptide based vaccine. Costimulatory molecule B7 can enhance antigenic peptide to induce antitumor responses.

Application of activated T lymphocytes induced by hepatocellular carcinoma rejection antigenic peptide-pulsed DCs in vivo and in vitro / 中国药理学通报

AIM To study if the activated T lymphocytes induced by hepatocellular carcinoma (HCC) rejection antigenic peptide SLIVHLNEV (mutant peptide of C met) pulsed dendritic cells (DCs) can provide protection against tumor challenge in nude mice HCC model and in vitro . METHODS SLIVHLNEV was eluted from HCC cell line HHCC by mild acid buffer and was identified by the mass spectrometry technique. SLIVHLNEV pulsed DCs isolated from HLA A2+ ruptured spleen were co cutured with isogeneic T lymphocytes. Cytotoxicity was tested using lactate dehydrogenase (LDH) release assay. The induced cytotoxic T lymphocytes (CTLs) were implanted into nude mice in order to protect them against transplanted HHCC tumor cells. The inhibition effect of CTLs on the growth of implanted tumor in nude mice was also observed. RESULTS The CTLs induced by SLIVHLNEV pulsed DCs had unique ability to kill HHCC tumor cells in vitro. They also protected the nude mice against the further challenge of HHCC tumor cells and inhibited the growth of implanted tumor. CONCLUSION DC based HCC rejection antigenic peptide SLIVHLNEV vaccine can induce powerful immune reaction both in nude mice HCC model and in vitro.