HPV16 L2E7 is a fusion protein used for therapeutical vaccine targeting HPV virus. To increase its expression in Escherichia coli, we optimized the codon usage of HPV16 l2e7 gene based on its codon usage bias. The optimized gene of HPV16 sl2e7 was cloned into three different vectors: pGEX-5X-1, pQE30, ET41a, and expressed in JM109, JM109 (DE3) and BL21 (DE3) lines separately. A high expression line was selected with pET41a vector in BL21 (DE3) cells. After optimization of the growth condition, including inoculation amount, IPTG concentration, induction time and temperature, the expression level of HPV16 L2E7 was increased from less than 10% to about 28% of total protein. HPV16 L2E7 protein was then purified from 15 L culture by means of SP Sepharose Fast Flow, Q Sepharose Fast Flow and Superdex 200 pg. After renaturing, HPV16 L2E7 protein with ≥ 95% purity was achieved, which was confirmed via SDS-PAGE gel and Western blotting. The combined use of purified HPV16 L2E7 and CpG helper has shown clear inhibition of tumor growth in mice injected with tumor cells, with six out of eight mice shown no sign of tumor. This study lays a solid foundation for a new pipeline of large-scale vaccine production.
Animals ; Capsid Proteins ; biosynthesis ; Codon ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; Genetic Vectors ; Human papillomavirus 16 ; Mice ; Neoplasms, Experimental ; prevention & control ; Oncogene Proteins, Viral ; biosynthesis ; Papillomavirus E7 Proteins ; biosynthesis ; Papillomavirus Vaccines ; therapeutic use ; Recombinant Fusion Proteins ; biosynthesis

ObjectiveTo construct one recombinant adenovirus AdE7E6 expressing HPV16 E6 and E7 fusion protein as candidate for HPV16 therapeutic vaccine.MethodsThe codon-optimized E6 and E7 gene,were fused to create one open reading frame,then inserted into adenovirus vector pCD316.A strain of recombinant adenovirus was constructed through homologous recombinant in 293 cells,and identified by PCR and Western blot.Finally,it was employed to study it's immunogenicity and the activity of the tumor growth regression.ResultsThe PCR result showed that E6E7 fusion gene had been integrated in recombinant Ad5 DNA.Western blot test confirmed that the E6E7 fusion protein was highly expressed in 293 cells infected with Ad5E7E6 recombinant adenovirus.The recombinant adenovirus elicited significant E7 specific CD8+ T lymphocyte response in vaccinated mice.These responses could completely prevent the TG-1 tumor cell bearing mice treated with AdE7E6 from developing into tumor.ConclusionThese results suggested that rAd5E7E6 could be a potent vaccine candidate for the treatment of HPV16-associated tumors and their precancerous transformations.

Monkeypox is a zoonotic disease caused by monkeypox virus. It is similar to human smallpox, although typically much less serious and limited to human-to-human transmission. Smallpox no longer occurs following its worldwide eradication in 1980, whereas monkeypox still occurs sporadically in parts of Africa. To respond to the outbreak of monkeypox correctly, we need to rely on effective method of virus detection. This review presents the development in monkeypox virus detection method in recent years to provide reference for the situation of potential virus spreading into our country and for preventing and acting correctly against bioterrorism.

OBJECTIVETo generate an HPV16 prophylactic vaccine candidate for cervical cancer.

METHODSHPV16 major capsid protein L1 gene and minor capsid protein L2 gene were amplified using PCR. These genes were mutated by PCR site-directed mutagenesis for removal of sequence motifs (TTTTTNT) which would cause transcription termination when expressed from a vaccinia virus early promoter, then inserted into a vaccinia virus expression vector. A strain replication-deficient recombinant vaccinia virus containing the mutant sequences was obtained through a homologous recombination and identified.

RESULTSThe nucleotide sequence remained the correct amino acid sequence of the L1 and L2 proteins after mutated. Full-length L1 and L2 proteins were generated in cells infected with the recombinant virus. The virus strain propagated at very low titer or could not reproduce in some kinds of cell derived from different human tissues.

CONCLUSIONSThe authors have generated a strain replication-deficient recombinant vaccinia virus expressing HPV16 L1 plus L2 proteins as an HPV16 prophylactic vaccine candidate for cervical cancer.

Capsid ; Capsid Proteins ; genetics ; Cell Line ; Cloning, Molecular ; Female ; Gene Expression ; Genetic Vectors ; Humans ; Oncogene Proteins, Viral ; genetics ; Papillomaviridae ; genetics ; physiology ; Papillomavirus Infections ; prevention & control ; Transfection ; Tumor Virus Infections ; prevention & control ; Uterine Cervical Neoplasms ; virology ; Vaccinia virus ; genetics ; physiology ; Virus Replication

OBJECTIVETo generate a candidate HPV16 vaccine for experimental and therapeutical use for cervical cancer.

METHODSThe mutants of HPV16 early E6 and E7 genes were inserted into a vaccinia virus expression vector. A strain of recombinant vaccinia virus was constructed through homologous recombination.

RESULTSShowed that the mutant E6 and E7 genes were located at TK gene region of vaccinia virus Tiantan strain in a head to head orientation under the control of early/late promoters, H6 and 7.5K respectively. Studies in mice indicated that VmE6E7 could elicit specific antibodies against E6 and E7, and retarded or prevented tumor development in a proportion of C57 BL/6 mice challenged by syngeneic HPV16E6 and E7 transformed tumor cells.

CONCLUSIONSThe success in constructing VmE6E7 provides a basis for the further development of HPV16 therapeutic vaccine.

Animals ; Female ; Genes, Viral ; genetics ; Genetic Vectors ; genetics ; immunology ; Mice ; Mice, Inbred C57BL ; Mutation ; Neoplasms, Experimental ; prevention & control ; Oncogene Proteins, Viral ; genetics ; Papillomaviridae ; genetics ; Papillomavirus E7 Proteins ; Recombination, Genetic ; Repressor Proteins ; Transfection ; Vaccinia virus ; genetics ; immunology

BACKGROUNDTo select the mutants of HPV type 16 E6 and E7 genes suitable for construction of vaccine for treatment of cervical cancer.

METHODSE6 and E7 genes were modified by site-directed mutagenesis. Several recombinant vaccina viruses were constructed by inserting the E6 or E7 mutants into the genome of vaccina virus Tiantan strain and employed to study their antigenicity.

RESULTSWestern blot assay showed that the E6 ?mutant? with substitution of Gly for Leu at amino acid site 50 and E7 mutant with substitution of Gly for Cys-24 and Glu-26 had no effect on their stability and antigenicity, but change of the Cys at position 91 of E7 dramatically reduced its stability and antigencity. Conclusion The results confirmed that the Zinc-finger structure at the E7 C-terminal? plays an important role in the integrity and stability of E7 protein.

Animals ; Antibodies, Viral ; biosynthesis ; Female ; Mice ; Mice, Inbred C57BL ; Mutagenesis, Insertional ; Oncogene Proteins, Viral ; genetics ; immunology ; Papillomaviridae ; genetics ; Papillomavirus E7 Proteins ; Repressor Proteins ; Vaccinia virus ; immunology ; Zinc Fingers

Objective: To detect the expression level of early and late protein of vaccinia virus and to preliminarily explore replication-defective mechanism of highly attenuated NTV strain of vaccinia virus Tiantan. Methods: We constructed prokaryotic expression vector, expressed and purified homologous early protein E3 and late protein A27 closely related to replication and prepared mouse polyclonal antiserum by immunizing mice with homologous proteins. Early and late protein expression levels of NTV were detected. Results: We have expressed and purified vaccinia virus proteins respectively in E. coli expression system and prepared homologous mouse polyclonal antiserum. Early protein E3 and late protein A27 could be highly efficient expression in NTV infected non-permissive Hela cells, while expression of late protein F17 was blocked detected by Western blot. Conclusions The expression limitation of late protein F17 may be an explanation for the replication-defective mechanism of NTV.

Objective To investigate the chemical constituents in the marine sponge, Spongia sp., collected from the Xisha Islands.Methods The pure chemical components from the petroleum ether extract of Spongia sp.were obtained by repeated column chromatography on silica gel,ODS,Sephadex LH-20 and semi-preparative HPLC.Their structures were determined by spectroscopic analysis and comparison with the reported data.The antifungal activity of those compounds was evaluated by dilution method.Results 9 compounds were isolated and identified,including smenodiol(1),smenospongorine(2),5-epi-smenospongorine(3),dictyoceratin C(4),epi-smenospongidine(5),dictyoceratin A(6),stigmasta-4,6,8(14),22-tetraen-3-one(7),3-oxo-4,6,8(14)-triunsaturated steroids(8),ergosta-4,6,8(14),22-tetraen-3-one(9).Conclusion Compounds 1~9 were isolated from the sponge of genus spongia for the first time.Compound 2、3、5 and 9 exhibited antifungal activities against Candida albicans,Trichophyton mentaqrophytes and Trichophyton rubrum with the MIC values of 12.5~25 μg/ml.

Objective To evaluate the virulence of NTV modified strain NTV-C7L in vitro and in vivo.Methods The non-replicative vaccinia virus modified strain NTV-C7L was constructed by homologous recombination,in order to evaluate the virulence of NTV-C7L by cell diffusion ability and virus replication,intranasal challenge,and scratch of tail of mice.Results The homologous recombination non-replicating vaccinia virus modified strain NTV-C7L was selected by blue-white screening,the purity of NTV-C7L was identified by PCR and Western blot,the C7L gene was successfully inserted and expressed;In human embryonic lung fibroblasts (MRC-5) as well as mouse embryonic fibroblasts (BALB/C3T3),the replication ability of NTV-C7L was recovered compared with that of NTV,but was lower than that of VTT;the result of the intranasal route with 106 PFU vaccinia virus showed that the animals infected with TTV had slightly greater weight loss than NTV or NTV-C7L(t =6.56,P＜0.001;t =5.73,P＜0.001).Compared with the NTV-C7L group,the weight loss of the NTV group was not statistically significant (t =0.597,P =0.081);the intranasal route with 107pFU vaccinia virus showed that the weight loss of the NTV group and NTV-C7L group was statistically significant compared with the TTV group (t =12.86,P＜0.001;t =10.71,P＜ 0.001).Compared with the NTV-C7L group,the weight loss of the NTV group was statistically significant(t =4.616,P＜0.001);the result of skin scratch indicated that mice scarified with TTV (106 PFU) formed more obvious skin redness,swelling and injury on the post infected 5 days,whereas only redness was detected after scarification with a much higher dose (107) of NTV or NTV-C7L.Conclusions The intracellular and mouse virulence of NTV modified strain NTV-C7L was recovered compared with NTV,but was significantly lower than that of TTV,which provided reference for optimization and application of vaccinia virus vector.

Objective:Using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) technology to edit Vaccinia virus (VACV) thymidine kinase (TK) Region for targeted recombination to establish an efficient vaccinia virus insertion recombination technology.Methods:We designed and synthesized guide RNAs(gRNA)targeting the TK region and then cloned individual gRNA into the PX458 vector that removes nuclear localization signals, and modified the original TK region recombinant plasmid. The gRNA and Cas9 co-expression plasmids were transfected into 293T cells separately to mediate the homologous recombination of vaccinia virus (VACV) and TK region recombination plasmid, and then the rate of viral recombination was evaluated by the appearance of blue and white spots.Results:The recombination efficiency mediated by the gRNA sequence designed and synthesized in this study is more than 1%, which is more than 10 times higher than the classical homologous recombination method .Conclusions:This study used CRISPR/Cas9 technology to establish a highly efficient recombinant vaccinia virus system, which provides technical support for pre-clinical research in vaccines or multivalent research of emerging infectious diseases, as well as tumor treatment.