The high expression of HPV31 and 52 L2 fusion protein in E. coli and detection of its immunogenicity / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To express HPV31 and 52 L2 fusion protein and detect its immunogenicity.</p><p><b>METHODS</b>According to the amino acid sequences of HPV31 and 52 L2 11-200AA published in the GenBank database, weartificially synthesized the HPV31 and 52 L2 fusion gene which was optimized according to Escherichia coli codon usage and encodes 11-200 amino acid of HPV31 and HPV52 L2, then cloned it into pET-9a vector. The HPV31 and 52 L2 fusion protein was expressed in Prokaryotic expression system and the mice were immunized with the fusion protein after purification. The immunogenicity was characterized in vaccinated mice.</p><p><b>RESULTS</b>HPV31 and 52 L2 fusion protein was highly expressed in E. coli, the amount of fusion protein is nearly 20% of the total bacterial protein. The purified fusion protein with aluminum adjuvant could induce specific high titer of IgG antibodies detected by ELISA, and also induce the neutralizing antibodies against pseudovirus of HPV31 and HPV52 and cross-neutralizing antibodies against pseudovirus of HPV45, 58, 16, 18.</p><p><b>CONCLUSION</b>HPV31 and 52 L2 fusion protein could induce neutralizing and cross-neutralizing antibodies against HPV pseudovirus. It provides laboratory basis for development of HPV L2 protein vaccine.</p>

High expression of HPV16L2N120E7E6 fusion protein in E. coli and its inhibitory effect on tumor growth in mice / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the high expression of HPV16L2N120E7E6 fusion protein by prokaryotic expression system, and evaluate its immunogenicity and antitumor efficacy in vaccinated mice.</p><p><b>METHODS</b>The HPV16L2N120E7E6 fusion gene, its codons were optimized to increase the expression of the protein, was constructed by overlap extension PCR and inserted into prokaryotic expression vector pET9a. Then the fusion protein was expressed by inducing with IPTG in E. coli strain BL21 (DE3) harboring with plasmid pETL2N120E7E6, and further detected by SDS-PAGE and Western-blot. Finally, the humoral and cellular immune responses were measured by ELISA and ELISPOT, respectively, in vaccinated mice with the purified HPV16L2N120E7E6 fusion protein, and the antitumor efficacy was assessed in mice using the TC-1 tumor challenge model.</p><p><b>RESULTS</b>The codon-optimized HPV16L2N120E7E6 fusion gene was highly expressed in E. coli strain BL21 (DE3) harboring with plasmid pETL2N120E7E6, and the amount of fusion protein was nearly 48.6% of the total bacterial protein. The purified fusion protein could induce high titer of specific antibody against L2, E7 and E6 in vaccinated mice. When accompanied with the adjuvant CpG, the fusion protein was able to elicit strong and moderate cellular immune responses in vaccinated mice against peptide HPV16E7(49-57) and peptide pools of HPV16E6, respectively. Furthermore, the tumor therapeutic experiment showed that HPV16L2N120E7E6 + CpG could prevent the tumor formation in 80.0% (8/10) vaccinated mice.</p><p><b>CONCLUSIONS</b>The data of this study suggest that HPV16L2N120E7E6 fusion protein could be a promising candidate vaccine for treatment of chronic HPV16 infection and post-operative adjuvant therapy for cervical cancer.</p>

Identification specific T lymphocyte epitopes on E6 protein of human papillomavirus type 18 in mice / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To identify specific T lymphocyte epitope on E6 protein of human papillomavirus type 18 in mice.</p><p><b>METHODS</b>Infection with one recombinant vaccinia virus rVVJ18 E7, E6 respectively in C57 BL/6 and BALB/c mice, specific cellular immune responses were detected by ELISPOT or intracellular cytokine stainings by using a series of overlapping synthetic peptides covering full length of the amino acid sequence of E6 and E7 proteins or various truncated peptides.</p><p><b>RESULTS</b>The rVVJ18 E7, E6 generated significant E6 specific T-cell immune responses in vaccinated mice. Mapping of the epitope of E6 revealed that the peptides E6(67-75 ( KCIDFYSRI) and E6(60-68) (IPHAAGHKC) presented respectively by C57BL/6 and BALB/c mice were the optimal peptides to activate E6-specific CD8+ T lymphocytes. However no positive cellular immune responses stimulated with various E7 peptides were detected by ELISPOT in immunized mice.</p><p><b>CONCLUSIONS</b>Two specific T lymphocyte epitopes were identified on E6 protein in C57BL/6 and BALB/c mice, which will provide the basis to evaluate cellular immune response elicited by HPV18 E6 protein based vaccine.</p>

The expression and preliminary evaluation of HPV6bL2deltaN360E7E6 fusion protein in E. coli for genital warts / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To express HPV6bL2deltaN360E7E6 fusion protein in E. coli and preliminarily evaluate its immune effect.</p><p><b>METHODS</b>Three HPV6b gene fragments, which were L2(1-360 bp), E7 and E6, were fused by overlapping PCR, then were inserted into a prokaryotic expression vector and expressed in E. coli. C57BL/6 mice were immunized with purified fusion protein plus Al (OH)3 and/or CpG adjuvants through intramuscular route, the cellular and humoral immune responses were detected by IFN-gamma ELISPOT and ELISA respectively.</p><p><b>RESULTS</b>Protein plus CpG adjuvant could induce the strongest cellular immune response to E7 and E6, high antibody titer against L2 could be detected in all immunized groups but there were no significant difference among these groups.</p><p><b>CONCLUSIONS</b>HPV6bL2deltaN360E7E6 gene was successfully cloned into pQE30 vector and expressed in E. coli, the fusion protein was also purified and proved that could induce strong cellular and humoral immune responses with appropriate adjuvant in C57BL/ 6 mice and could be used for future research.</p>

Construction of recombinant vaccinia virus expressing HPV18E7E6 fusion proteins and detection of its immunogenicity in mice / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct one recombinant vaccinia virus expressing the HPV18 E6 and E7 fusion proteins as HPV18 therapeutic vaccine candidate, and test its immunogenicity.</p><p><b>METHODS</b>The fusion E7E6 genes were synthesized and mutated to inactivate their oncogenic potential, and inserted into a vaccinia virus plasmid vector to construct one recombinant vaccinia virus. Finally its immunogenicity was characterized in immunized mice.</p><p><b>RESULTS</b>One recombinant vaccinia virus expressing HPV18 E7E6 fusion proteins was constructed. Sequencing results of PCR products and Western blot tests showed that the E7E6 fusion genes were correct and expressed in CEF cells infected with the recombinant vaccinia virus. The specific antibodies against E6 and E7 proteins were elicited, however no positive responses were detected by ELISPOT in immunized mice.</p><p><b>CONCLUSIONS</b>One recombinant vaccinia virus expressing HPV18 E7E6 fusion proteins was generated and elicited specific antibodies against E6 and E7 proteins, but detected no positive cellular immune responses in immunized mice, which will provide the basis to develop the different animal model for examining the cellular immune responses of HPV18E6 and E7 proteins.</p>

Prokaryotic expression and purification of human papillomavirus type 11 L2E7 fusion protein vaccine and its immunnogenicity / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct the Escherichia coli (E. coli) prokaryotic expression system pET9aHPV11L2E7, purify the fusion protein L2E7 and study the immunnogenicity of the protein.</p><p><b>METHODS</b>The HPV11 L2, E7 coding region was amplified from condyloma acuminata tissue specimen by PCR. The recombinant plazmid pET9aHPV11L2E7 was established and sequenced. Fusion protein L2E7 (553 amino acids) was expressed in host strain BL21 (DE3plus) by IPTG inducing and identified by using SDS-PAGE and Western blotting. Then L2E7 protein purified with CM column was inoculated to Balb/c mice and its cell-mediated and humoral immunnogenicity was assessed by IFN-gamma enzyme-linked immunospot (ELISPOT) and enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>The E. coli prokaryotic expression system pET9aHPV11L2E7 was established and the purified fusion protein L2E7 was obtained successfully. The mice in vivo experiment indicated that the purified protein L2E7 could induce HPV11E7 specific cell-mediated immune responses and high level HPV L2E7 antibody was detected in serum.</p><p><b>CONCLUSION</b>The purified fusion protein L2E7 could induce specific cell-mediated and humoral immune responses. It can be used as a candidate of genital wart immune therapeutic vaccine.</p>

Highly efficient expression of codon-optimized human papillomavirus 16 L2E7 gene in Escherichia coli / 中国医学科学院学报

<p><b>OBJECTIVE</b>To enhance the expression level of human papillomavirus (HPV) 16 L2E7 in Escherichia coli (E. coli), in aim of providing high-level expression of HPV16 L2E7 strain for pre-clinical high-throughout production.</p><p><b>METHODS</b>The whole L2E7 gene was optimized by software of Synthetic Gene Designer, reflecting E. coli codon usage. Two parts of codon-optimized gene were cloned into pET9a vector step by step. The positive clone, which was sequenced to be corrected, was transfected to BL21 (DE3+) via isopropyl-beta-D-thiogalactoside (IPTG) induction. They produced the HPV16 L2E7 fusion protein, which was further detected by SDS-PAGE and Western blot. The induction temperature, induction time, and IPTG concentration were also optimized by a series of experiments. Further purification modes of this protein were also explored.</p><p><b>RESULTS</b>Codon-optimized HPV16 L2E7 was highly expressed in E. coli. The target protein accounted for nearly 60% of the total cell extract.</p><p><b>CONCLUSION</b>High-level expression of HPV16 L2E7 was successfully constructed.</p>

Expression of the human papillomavirus type 16L/E7 fusion protein in E. coli and observation of its immunogenicity in mice / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>Many epidemiological and experimental evidences prove that cervical cancers are strongly associated with genital high-risk types of human papillomavirus (HPV). HPV16 is present in 50% of the tumor specimens. Thus, it is important to develop vaccines against HPV16 and cervical cancer. The authors studied the expression of the HPV16 L1DeltaCE7N fusion protein in E. coli and observed its immunogenicity.</p><p><b>METHODS</b>The fragment of HPV16 L1DeltaC gene and the E7N gene were amplified by PCR separately; the fusion gene named L1DeltaCE7N was generated by fusing E7N to the C terminal of L1DeltaC then the chimeric gene was cloned into prokaryotic expression vector pGEX-2T and expressed in E. coli strain JM109. The L1DeltaCE7N protein expressed were detected by Western blot. Finally its immunogenicity was characterized in immunized mice.</p><p><b>RESULTS</b>It was proved that the sequence and open reading frame of fusion gene L1DeltaE7N was correct by sequencing; SDA-PAGE gel analysis showed that HPV16 L1/E7 fusion protein was highly expressed in E. coli; the protein was expressed as soluble form and the molecular weight was about 85 x 10(3). The fusion protein could be purified by affinity chromatography and gel filtration. The ELISA result indicated that L1/E7 could elicit specific antibodies against L1 and E7 in immunized mice. In vivo tumor protection test indicated that tumor formation was retarded or prevented in the mice after vaccination with L1/E7, when C57 BL/6 mice were challenged by syngeneic HVP16E6 and E7 transformed tumor cells.</p><p><b>CONCLUSION</b>HPV16L1/E7 fusion protein was expressed in E. coli, it can be a candidate for prophylactic and therapeutic vaccine for HPV16-associated infection and tumors.</p>

Construction and identification of non-replication recombinant vaccinia virus co-expressing human papillomavirus type 16 L1/L2/E6/E7 proteins / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To generate a human papillomavirus (HPV16) prophylactic and therapeutic vaccine candidate for cervical cancer.</p><p><b>METHODS</b>HPV16 major capsid protein L1 gene/minor capsid protein L2 gene and HPV16 early E6/E7 genes were inserted into a vaccinia virus expression vector. A strain of non-recombinant vaccinia virus containing the sequences was obtained through a homologous recombination and identified.</p><p><b>RESULTS</b>DNA hybridization confirmed that the HPV16L1/L2/E6/E7 genes were integrated into vaccinia virus DNA. Western Blot result showed that full-length L1/L2/E6/E7 proteins were co-expressed in CEF cells infected with the recombinant virus.</p><p><b>CONCLUSION</b>NTVJE6E7CKL1L2 could be taken as a candidate of prophylactic and therapeutic vaccine for HPV-associated tumors and their precancerous transformations.</p>

Non-replicating recombinant vaccinia virus expressing HPV16 E6 and E7 proteins elicits anti-tumor immunity in mice / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the anti-tumor immunity of the non-replicating recombinant vaccinia virus expressing HPV16 E6 and E7 proteins.</p><p><b>METHODS</b>C57BL/6 mice were immunized by non-replicating recombinant vaccinia virus (NTVJmE6E7), and then specific CTLs were determined. Immune protection effects were evaluated by challenges of different doses of TC-1 tumor cells. Immunotherapeutic effects in form of recurrence were evaluated on the tumor-removed mice.</p><p><b>RESULTS</b>Mice immunized by NTVJmE6E7 could generate TC-1 cell specific cytotoxic T lymphocyte (CTL). Mice boosted with NTVJmE6E7 could tolerate the challenge of 1 x 10(4) TC-1 cells. NTVJmE6E7 could effectively prevent the tumor recurrence in the tumor-removed mice.</p><p><b>CONCLUSION</b>NTVJmE6E7 can be taken as a candidate of therapeutic vaccine for HPV-associated tumors and their precursor lesions.</p>