Culture of human sapovirus GⅠ.1 in vitro and preparation of polyclonal antibody against capsid protein VP1 / 中国生物制品学杂志

@#Objective To culture human sapovirus(HuSaV) GⅠ.1 in vitro and prepare polyclonal antibody against the capsid protein VP1.Methods HuSaV GⅠ.1 positive stool specimens preserved in diarrhea department of National Institute for Viral Disease Control and Prevention were inoculated with HuTu-80 cells supplemented with different bile acid salts[glycine chenodeoxycholic acid(GCDCA) and glycine cholic acid(GCA)],and the infection,proliferation and passage of the virus were determined by PCR and RT-qPCR.The VP1 gene was amplified by PCR and cloned into prokaryotic expression vector pGEX-6P-1.The constructed recombinant expression plasmid pGEX-6P-1-VP1 was transformed into E.coli BL21(DE3) and induced by IPTG.Two female New Zealand white rabbits were immunized with the purified recombinant VP1 protein for 4 times.The blood samples were collected 18,28,38 and 48 d after immunization,and the serum titers were detected by ELISA.Results HuTu-80 cells were effectively infected by HuSaV GⅠ.1 in the presence of bile acid salt GCA,and the proliferated virus were stably and continuously transmitted for three generations in HuTu-80 cells.The expressed recom-binant GST-VP1 protein showed a relative molecular mass of about 86 000,and about 60 000 after purification(GST tag excision).The titer of polyclonal antibody against HuSaV VP1 protein was over 1:12 800.Conclusion HuSaV was successfully isolated and cultured in vitro using HuTu-80 cells supplemented with bile acid salt,and polyclonal antibody with high titer against HuSaV VP1 protein was prepared,which laid a foundation of in-depth research of HuSaV identification,infection and pathogenesis.

Prokaryotic expression and immunogenicity of hepatitis A virus capsid proteins VP1 and VP3 / 中国生物制品学杂志

@#Objective To express the capsid proteins VP1 and VP3 of hepatitis A virus(HAV) in prokaryotic cells and evaluate their immunogenicity.Methods VP1 and VP3 gene fragments were amplified by PCR,cloned into vector pETG28a to construct recombinant expression plasmids pET-G28a-VP1 and pET-G28a-VP3,which were transformed into competent E.coli BL21(DE3),induced by IPTG,and then analyzed by 12% SDS-PAGE.The target protein was purified by ion exchange chromatography,renatured and combined with several adjuvants to immunize mice.The mice were divided into VP1-MF59,VP1-AH,VP1-AP,VP1-AP-10CpG,VP1-AP-50CpG,VP3-AP,VP3-VP1-AP and PBS control groups,five for each group.Serum IgG antibody titers of mice in various groups were detected by ELISA,and serum neutralizing antibody titers of mice in VP1-AP,VP3-VP1-AP and PBS control groups were detected by rapid fluorescence focus immunosuppression experiment.Results Colony PCR and sequencing showed that the recombinant plasmids pET-G28a-VP1 and pET-G28a-VP3were constructed correctly.The recombinant proteins VP1 and VP3,with relative molecular masses of about 37 000 and26 000 respectively,mainly existed in the form of inclusion bodies,the expression levels were 18.6% and 32.4%,and the purity was 86.3% and 84.7%,respectively.The recombinant proteins VP1 and VP3 reacted specifically with rabbit anti-HAV antiserum and mouse anti-HAV-VP3 antiserum respectively.The serum IgG antibody titer of mice in VP1-AP-50CpG group was significantly higher than that in VP1-AP group(q=22.05,P <0.01),and the serum IgG antibody titer of mice in VP3-VP1-AP group was significantly higher than that in VP1-AP group and VP3-AP group(q=22.05 and 22.49 respectively,each P <0.01).Compared with PBS control group,the serum neutralizing antibody titer of mice in VP1-AP and VP3-VP1-AP group increased significantly(q=7.79 and 25.11 respectively,P<0.01) Conclusion Prokaryotic HAV capsid proteins VP1 and VP3 showed high purity and good immunogenicity,and the addition of CpG in the preparation was beneficial to enhance the immunogenicity of antigens.This study laid a foundation of the development of HAV recombinant subunit vaccine.

Novel PF74-like small molecules targeting the HIV-1 capsid protein: Balance of potency and metabolic stability

Of all known small molecules targeting human immunodeficiency virus (HIV) capsid protein (CA), PF74 represents by far the best characterized chemotype, due to its ability to confer antiviral phenotypes in both early and late phases of viral replication. However, the prohibitively low metabolic stability renders PF74 a poor antiviral lead. We report herein our medicinal chemistry efforts toward identifying novel and metabolically stable small molecules targeting the PF74 binding site. Specifically, we replaced the inter-domain-interacting, electron-rich indole ring of PF74 with less electron-rich isosteres, including imidazolidine-2,4-dione, pyrimidine-2,4-dione, and benzamide, and identified four potent antiviral compounds (

Advances in research on HBV inhibitors based on new targets (1): capsid protein inhibitors / 药学学报

Hepatitis B virus (HBV) capsid protein plays an important role in the life cycle, thus becoming an ideal target for drug design. Capsid protein inhibitors can exert a synergistic antiviral effect with nucleoside drugs by inhibiting the replication of HBV. This paper reviews the research progress of capsid protein inhibitors with different structural types from the perspective of medicinal chemistry.

Caracterización de la proteína no capsidal 3D, del virus de la fiebre aftosa y producción de anticuerpos policlonales / Characterization of the non-capsided 3d protein, of the foot-and-mouth disease virus and the production of polyclonal antibodies

Las proteínas no capsidales del virus de la fiebre aftosa se utilizan como marcadoras en la evaluación de animales que han estado en contacto con el virus, a diferencia de los inmunizados, ya que la vacuna no debe tener estas proteínas, por lo tanto los animales no deben presentar anticuerpos contra ellas. El objetivo de esta investigación fue la caracterización de la proteína no capsidal 3D y la producción de anticuerpos policlonales in vivo. La proteína se purificó del cultivo de virus inactivo, por cromatografía de intercambio iónico. La elución de los picos fue sometida a electroforesis uni-bidimensional; demostrándose un alto grado de pureza (>90%) en el pico tres, donde se identifico la proteína 3D, por la técnica de MALDI-TOF y electroespray de trampa iónica. La proteína purificada, se inoculó en cabras y el suero hiperinmune fue precipitado y sometido a cromatografía de afinidad para la obtención de inmunoglobulinas; la reacción inmunitaria se confirmó por medio de inmunodifusión y Western blot. El proceso de purificación demostró ser eficiente y útil para la obtención de anticuerpos específicos, los cuales tendrán utilidad en la elaboración de un ensayo inmunoenzimático que mida la pureza de la vacuna frente al contenido de estas proteínas.

The noncapsid proteins of the foot and mouth disease are used as markers in the evaluation of animals that have been in contact with the virus, to discriminated the immunized animals, because the vaccine should not have these proteins, therefore animals should not present antibodies against them. The aim of this investigation was the characterization of the 3D non-capsid protein and the production of polyclonal antibodies in vivo. The protein was purified from the culture of inactivated virus, by ion exchange chromatography. The elution of the peaks were submit an one-two-dimensional electrophoresis; Demonstrated a high degree of purity (> 90%) in peak three, where the 3D protein was identified, by the MALDI-TOF technique and ion trap electrospray. The purified protein, inoculated in goats and the hyperimmune serum, was precipitate out and submitted to affinity chromatography to obtain immunoglobulins; the immune reaction was confirmed by means of immunodiffusion and Western blot. The purification process proved to be efficient and useful for obtaining specific antibodies, which will be useful in the preparation of an immunoenzymatic assay that measures the purity of the vaccine against to the content of these proteins.

Clonación y expresión en Escherichia coli de un gen L1 completo del virus del papiloma humano 18 aislado de una paciente cubana y variantes delecionadas / Cloning and expression in Escherichia coli of the full-length and deletion variants of a human papillomavirus 18 L1 gene isolated from a Cuban patients

Introduction: Human papillomavirus (HPV) vaccines are based on the L1 major capsid protein. Objectives: To clone the HPV-18 L1 gene from a Cuban female HPV-18-infected patient and to express the full-length and deletion variants of the cloned HPV-18 L1 gene in Escherichia coli. Methods: The full-length HPV-18 L1 gene was PCR-amplified from total DNA isolated from a Cuban patient, cloned and finally subcloned into the E. coli expression vector pET26b. Three deletion mutants were constructed, which encode truncated proteins lacking 30 amino acids at the C-terminus in combination with 5, 6 or none deleted residue at the N-terminus. Production of L1 proteins in E. coli BL21(DE3) and E. coli SHuffle T7 was assessed by SDS-PAGE and Western blotting. Results: The cloned HPV-18 L1 gene was 99.9 por ciento similar to the African variant EF202152 and probably shares a common origin with the B lineage of genotype 18. The three truncated variants of HPV-18 L1 were produced at higher levels than the full-length HPV-18 L1 protein, attaining higher levels in E. coli BL21(DE3) and higher solubility in E. coli SHuffle. The C-terminus-only truncated variant, L1∆C30, was produced at similar levels to the HPV-18 L1s truncated at both termini. E. coli SHuffle produced about three times more amounts of L1∆C30 when grown under autoinduction conditions with respect to conventional induction and thus, amounts were comparable to those obtained in E. coli BL21(DE3) under conventional induction. Conclusions: Truncation of thirty amino acid residues at the carboxy-terminus of the HPV-18 L1 made a major contribution to the production and solubility of this wild-type protein in E. coli. This is the first report about soluble production of HPV-18 L1 protein in an E. coli SHuffle strain. However, higher amounts of L1 are needed to scale-up its production for developing an HPV vaccine candidate(AU)

Introducción: Las vacunas contra el virus del papiloma humano (VPH) se fundamentan en la proteína principal de la cápsida L1. Objetivo: Clonar el gen L1 del VPH-18 a partir de una paciente cubana infectada con VPH-18 y expresar las variantes de longitud completa y delecionadas del gen L1 del VPH-18 en Escherichia coli. Métodos: El gen L1 del VPH-18 de longitud completa se amplificó por PCR a partir de ADN total aislado de un paciente cubana, se clonó y finalmente se subclonó en el vector de expresión de E. coli pET26b. Se construyeron tres mutantes de deleción, que codifican para proteínas truncadas que carecen de 30 aminoácidos por el extremo carboxilo, en combinación con 5, 6 o ningún residuo delecionado por el extremo amino. La producción de las proteínas L1 en E. coli BL21(DE3) y E. coli SHuffle T7 se evaluó mediante SDS-PAGE y Western blot. Resultados: El gen L1 del VPH-18 clonado fue 99.9 percent similar a la variante africana EF202152 y probablemente comparte un origen común con el linaje B del genotipo 18. Las tres variantes truncadas de la proteína L1 del VPH-18 se produjeron a mayores niveles que la proteína L1 del VPH-18 de longitud completa, alcanzando mayores niveles en E. coli BL21(DE3) y mayor solubilidad en E. coli SHuffle. La variante truncada solo por el extremo carboxilo, L1(C30, se produjo a niveles similares a las proteínas L1 del VPH-18 truncadas por ambos extremos. E. coli SHuffle produjo aproximadamente tres veces más cantidades de L1(C30 cuando creció en condiciones de autoinducción, con respecto a la inducción convencional y, por ende, las cantidades fueron comparables a las obtenidas por E. coli BL21(DE3) bajo inducción convencional. Conclusiones: La truncación de treinta residuos de aminoácidos por el extremo carboxilo de la proteína L1 del VPH-18 tuvo una importante contribución a la producción y solubilidad de la proteína L1 nativa en E. coli. Este es el primer informe sobre la producción soluble de la proteína L1 del VPH-18 en una cepa de E. coli SHuffle. Sin embargo, se necesitan mayores cantidades de la proteína L1 para escalar su producción para desarrollar un candidato vacunal contra el VPH(AU)

Advance in human papillomavirus major capsid protein L1-based vaccines / 中华微生物学和免疫学杂志

L1 is the major capsid protein of human papillomavirus (HPV) encoded by late gene 1. Based on the fact that L1 can self-assemble into virus like particle (VLP) with good immunogenicity, it has aroused wide concern in studying the pathogenesis of and vaccines against HPV. Nevertheless, there are a few limitations of present L1-based HPV vaccines. For instance, low expression of the protein and the complexity of purification result in the relatively low yield of vaccines. Type-specific antibody induced by L1 also results in the unsatisfactory cross-protection rate. Furthermore, there is no reported therapeutic effect against HPV-related diseases because of its undefined role in virus eliminating. This review focused on the structure, immunogenicity and role in immune response of L1 and the development of and latest progress in HPV vaccines.

Advance in human papillomavirus major capsid protein L1-based vaccines / 中华微生物学和免疫学杂志

L1 is the major capsid protein of human papillomavirus (HPV) encoded by late gene 1. Based on the fact that L1 can self-assemble into virus like particle (VLP) with good immunogenicity, it has aroused wide concern in studying the pathogenesis of and vaccines against HPV. Nevertheless, there are a few limitations of present L1-based HPV vaccines. For instance, low expression of the protein and the com-plexity of purification result in the relatively low yield of vaccines. Type-specific antibody induced by L1 also results in the unsatisfactory cross-protection rate. Furthermore, there is no reported therapeutic effect against HPV-related diseases because of its undefined role in virus eliminating. This review focused on the struc-ture, immunogenicity and role in immune response of L1 and the development of and latest progress in HPV vaccines.

Progress of screening methods in vitro for HIV-1 capsid protein inhibitors / 中国药理学通报

Human immunodeficiency virus type 1 (HIV-1) infection is highly dependent on its conical fullerene capsid, which is composed of approximately 1 500 capsid proteins. In recent years, capsid protein has been recognized as an ideal target for design and screening drugs for AIDS therapy. Screening methods are the key to develop HIV-1 capsid inhibitors. A variety of screening approaches for HIV-1 capsid protein inhibitor, which have been reported in the literature are reviewed in the article.

Advances in HBV capsid protein assembly modulators / 中国药理学通报

Hepatitis B virus (HBV) belongs to the Hepadnaviridae family. HBV infection may lead to acute and chronic hepatitis, which has become a global health issue. HBV capsid protein assembly modulators play an important role in multiple stages of HBV replication, such as encapsidation of pgRNA and synthesis of HBV DNA, thus becoming a new hotspot in the research and development of hepatitis B drugs. This paper reviews the research progress of HBV capsid protein assembly modulators.

The expression characteristics and co-localization of HIV capsid protein p24 and TRIM22 in U-251 glioma cells / 西安交通大学学报(医学版)

Objective: To observe the expression characteristics and co-localization of exogenous TRIM22 and HIV capsid protein p24 in glioma cells. Methods: The vectors of pEGFP-N3-TRIM22 or pDsRed1-p24 were transfected into U-251 glioma cells respectively to examine the expression of TRIM22-EGFP or p24-DsRed1 by confocal microscopy. Moreover, we used a confocal z-stacking program to achieve series of optical sections and to rebuild 3-D images by ImageJ 1.50i software to detect the expression characteristics of p24-DsRed1 in U-251 cells. In the end, the vectors of pEGFP-N3-TRIM22 and pDsRed1-p24 were co-transfected into U-251 cells to detect the co-localization between TRIM22 and p24 by confocal microscopy. Results: Confocal microscopy results showed that TRIM22-EGFP or p24-Dsred1 was localized to the cell body as well as to protuberance in U-251 cells, and 3D structural reconstruction showed that p24-Dsred1 could be transferred to foot processes of U-251 cells. Simultaneously, confocal microscopy results also showed that TRIM22 and p24 could be co-localized and their combination could be released by budding in U-251 cells co-transfected with pEGFP-N3-TRIM22 and pDsRed1-p24. Conclusion: TRIM22 co-localized to HIV capsid protein p24 and their combination can be released by budding in glioma cells.

Enhanced protective immune response to PCV2 adenovirus vaccine by fusion expression of Cap protein with InvC in pigs

The major immunogenic protein capsid (Cap) of porcine circovirus type 2 (PCV2) is critical to induce neutralizing antibodies and protective immune response against PCV2 infection. This study was conducted to investigate the immune response of recombinant adenovirus expressing PCV2b Cap and C-terminal domain of Yersinia pseudotuberculosis invasin (Cap-InvC) fusion protein in pigs. The recombinant adenovirus rAd-Cap-InvC, rAd-Cap and rAd were generated and used to immunize pigs. The phosphate-buffered saline was used as negative control. The specific antibodies levels in rAd-Cap-InvC and ZJ/C-strain vaccine groups were higher than that of rAd-Cap group (p < 0.05), and the neutralization antibody titer in rAd-Cap-InvC group was significantly higher than those of other groups during 21–42 days post-immunization (DPI). Moreover, lymphocyte proliferative level, interferon-γ and interleukin-13 levels in rAd-Cap-InvC group were increased compared to rAd-Cap group (p < 0.05). After virulent challenge, viruses were not detected from the blood samples in rAd-Cap-InvC and ZJ/C-strain vaccine groups after 49 DPI. And the respiratory symptom, rectal temperature, lung lesion and lymph node lesion were minimal and similar in the ZJ/C-strain and rAd-Cap-InVC groups. In conclusion, our results demonstrated that rAd-Cap-InvC was more efficiently to stimulate the production of antibody and protect pigs from PCV2 infection. We inferred that InvC is a good candidate gene for further development and application of PCV2 genetic engineering vaccine.

Structure analysis of capsid protein of Porcine circovirus type 2 from pigs with systemic disease

Abstract Economic losses with high mortality rate associated with Porcine circovirus type 2 (PCV2) is reported worldwide. PCV2 commercial vaccine was introduced in 2006 in U.S. and in 2008 in Brazil. Although PCV2 vaccines have been widely used, cases of PCV2 systemic disease have been reported in the last years. Eleven nursery or fattening pigs suffering from PCV2 systemic disease were selected from eight PCV2-vaccinated farms with historical records of PCV2 systemic disease in Southern Brazil. PCV2 genomes were amplified and sequenced from lymph node samples of selected pigs. The comparison among the ORF2 amino acid sequences of PCV2 isolates revealed three amino acid substitutions in the positions F57I, N178S and A190T, respectively. Using molecular modeling, a structural model for the capsid protein of PCV2 was built. Afterwards, the mutated residues positions were identified in the model. The structural analysis of the mutated residues showed that the external residue 190 is close to an important predicted region for antibodies recognition. Therefore, changes in the viral protein conformation might lead to an inefficient antibody binding and this could be a relevant mechanism underlying the recent vaccine failures observed in swine farms in Brazil.

Neutralizing epitopes and intragenotypic variants of human papillomavirus 16 (HPV16) / 中华微生物学和免疫学杂志

Cervical cancer is the second most common cancer in women worldwide. It is clear that persistent infection of high-risk human papillomaviruses (HR-HPVs) is the main cause for this disease. Among the several HPV types associated with carcinoma, HPV-16 is the most prevalent type and present in about 50% of tumor specimens. The major capsid protein (L1) of HPV can self-assemble into virus-like particles (VLPs) with immunogenicity similar to infectious virions. Neutralizing epitopes are the structural basis of the current prophylactic HPV vaccines. The efficacy of HPV vaccines is critically dependent upon the integrity of type-specific neutralizing epitopes. Recently, considerable headway has been made in studying the epitopes of HPV16 based on neutralizing antibodies. Notably, more and more HPV16 variants have appeared along with increasing immune pressure. To study the phenotypic variations in HPV16 L1 protein, 1 204 naturally occurring sequences were analyzed and a phylogenetic tree was then constructed including four clades. Moreover, after compared the aforementioned sequences with the 114K reference sequence, eight "hot mutation sites" , six "specialized mutation sites" and 20 "epitope-related mutation sites" were found. Generally, sera raised against VLPs can neutralize the corresponding HPV types, but not other types. However, it is not known whether intragenotypic variants of human papillomavirus type 16 (HPV-16) can be neutralized by sera vaccinated with a single variant VLPs. It is, therefore, imperative to understand the neutralizing epitopes and intragenotypic variants of HPV-16 for the production of prophylactic vaccines with high potency and broad coverage.

Study progress in capsid protein VP1 of enterovirus 71 / 中华实用儿科临床杂志

Human enterovirus 71 (EV71) is one of the major causative agent of hand,foot and mouth disease (HFMD) in infant.Clinical studies find that EV71 infection can cause a variety of clinical manifestations,from mild HFMD to fatal neurogenic pulmonary edema and even death,but the reasons remain unclear.The capsid protein VP1 of EV71 plays a key role in the processes of viral recognizing,binding and entering into the targeted cells and viral particles assembling.VP1 variation is a major determinant to EV71 fitness and immunogenicity.This study reviews the research progress of the structure,functions and associated antiviral vaccines and drugs of VP1,which further provides a theoretical basis for developing new and more effective antiviral vaccine and drugs.

Review for progress and current status of human papillomavirus prophylactic vaccine / 中国生化药物杂志

Persistant infection of high-risk human papillomavirus (HPV) is the primary cause leading to cervical cancer, which is ranked as second cancer threatening the health of women following breast cancer.Development of HPV vaccine is very important because there is no effective therapeutics for cervical cancer.Three currently licensed HPV vaccines based on major capsid protein L1 in the foreign market confered good safety and efficacy in clinical trials, but the current price is expensive due to high cost, which limits the wide application in developing countries.So far, the vaccines have not been launced in China market.Here, we review the progress and the current status of the HPV vaccine, which will attract the readers’ interest on the forthcoming emergence of HPV vaccine in China.

The physical states of HPV DNA and expression of human papillomavirus late capsid protein 1 in HepG2 cells / 基础医学与临床

Objective To find out the physical state of the human papillomavirus ( HPV) genome in hepatoma cell line HepG2 cells and the regulation of HPV late capsid protein 1 ( L1) expression and to explore the nature of the cytoryctes in HepG2 cells.Methods E2 and E6 in HPV18 were detected by PCR to evaluate the physical state of HPV18 genome .HepG2 L1 expression was detected by ELISA , light microscropy and electron microscrope immu-nohistochemistry assays , Western blot assay using HPV L 1 mice monoclonal antibody .L1 mRNA in HepG2 cells was detected by reverse transcriptional PCR ( RT-PCR) .Results PCR assay displayed that HPV DNA was inte-grated with HepG2 genome.ELISA assay showed that HPV L1 was present in lysate of HepG2 cells.Light micros-cropy demonstrated strong positive reaction in HepG2 cells.In microscopy, in the cytoplasm of partial HepG2 cells, there were lumpish cytorrhyctes materials which consists of very small and uniform particles and these parti -cles were marked by HPV L1 antibody labeled by colloidal gold .Western blot analysis showed a band at 56 ku dis-trict and it was L1 specific strap which demonstrated HPV 18 L1 was present in HepG2 cells.RT-PCR assay demon-strated the presence of L1 mRNA in HepG2 cells.Conclusions HepG2 cells are HPV18-positive HPV DNA ge-nome is integrated with HepG2 cells.HepG2 cells can express L1.The cytorrhyctes in HepG2 cells are composed of HPV18 L1 indicating that L1 can be expressed in HepG2.

Effect of boningmycin on expression of capsid protein L1 and serum immune factors in cervical HPV infection patients / 中国生化药物杂志

Objective To investigate the effect of boningmycin on expression of capsid protein L1 and serum immune factors in cervical HPV infection patients.Methods 60 patients with cervical HPV infection were divided into experimental group and control group, 30 cases in each group.The control group were given recombinant human interferon α2b suppository, 100 000 IU, once every other day, and the experiment group were given boningmycin 300 mg intramuscular injection,once every other day.Treatment of 10 times for a course,2 consecutive courses After treatment, the HPV capsid protein L1, serum TNF-α, IL-2, IFN-αand HPV were compared.Results Compared with control group after treatment, the percentage of HPV capsid protein L1 increased ( P <0.05 ) , serum TNF-α, IFN-αlevels increased ( P <0.05 ) , IL-2 decreased ( P <0.05 ) , and HPV positive rate decreased( P <0.05 ).Conclusion Boningmycin could reduce the percentage of capsid protein L1 in patients with cervical human papilloma virus infection, elevate serum TNF-αand IFN-αlevels, reduce the level of IL-2 and HPV positive rate.

Expressions of human papillomavirus L1 and human telomerase RNA component gene in high-risk types of human papillomavirus positive cervical exfoliated cells / 肿瘤研究与临床

Objective To evaluate the significance of human papillomavirus L1 capsid protein (HPVL1) and human telomerase RNA component (hTERC) gene in the cytologic specimens of cervix which was infected by the high-risk types of human papillomavirus (HR-HPV),and to expose their relationship with cervical lesions.Methods The fluorescence signal of cytologic samples of cervix were detected by interphase FISH in chromosome enumeration double-color DNA probes TERC.The expression of HPVL1 capsid protein was detected by MaxVision immunohistochemistry method.300 samples were analyzed with HR-HPV positive from the cervical biopsy.The diagnoses as normal or chronic inflammation (n =45),cervical intraepithelial lesions Ⅰ grade (CIN Ⅰ,n =95),CIN Ⅱ (n =58),CIN Ⅲ (n =64),and squamous cervical cancer (SCC,n =37).Results The percentage of HPVL1 positive rates in normal or chronic inflammation,CIN Ⅰ,CIN Ⅱ,CIN Ⅲ and SCC groups were 58.70 ％ (27/46),63.16 ％ (60/95),37.93 ％ (22/58),10.94 ％ (7/64) and 0 (0/37),respectively.The percentage of HPVL1 decreased along with the increase of severity of the cervical intraepithelial lesions.Genomic amplification of hTERC positive rates in normal or chronic inflammation,CIN Ⅰ,CIN Ⅱ,CIN Ⅲ and SCC groups were 6.52 ％ (3/46),11.58 ％ (11/95),51.72 ％ (30/58),85.94 ％ (55/64) and 100.00 ％ (37/37),respectively.The percentage of hTERC increased along with the severity of the cervical intraepithelial lesions (rs =0.302,P ＜ 0.01).The percentage of HPVL+/hTERC-was 57.89 ％ in CIN Ⅰ group and 4.69 ％ in CIN Ⅲ group.The percentage of HPVL-/hTERC+ was 6.32 ％ in CIN Ⅰ group and 79.69 ％ in CIN Ⅲ group.Conclusion The detection of HPVL1 and hTERC are important for assisting cervical lesions screening and monitoring of disease progression in the HR-HPV positive cytologic specimens.

L1 C-terminal common sequence polypeptide antibody in detecting human papillomavirus in liquid-based cervical cytology specimens / 第二军医大学学报

Objective To determine whether the polypeptide antibody induced by human papillomavirus (HPV)L1 C-terminal conserved sequence polypeptide can be used to detect HPV in cervical exfoliated cell specimens. Methods Cervical exfoliated cell specimens were collected and divided into two groups: one was subjected to nested polymerase chain reaction (Nest-PCR) for detection of HPV DNA, and the other was subjected to sandwich enzyme-linked immunosorbent assay (ELISA), in which rabbit anti-polypeptide purified antibody and mice anti-polypeptide antiserum were used to determine HPV L1 in specimens. The detection rates of HPV were compared between the two groups. Part of the specimens were used for immunocytochemistry detection. Results Nest-PCR with primer MY09/11 detected 30 HPV DNA positive specimens out of a total of 296 ones, with a detection rate of 11.15%. Then the HPV DNA negative specimens were further amplified with primer GP5+/6, and 51 (21.34%) more positive cases were identified, making the total detection rate of Nest-PCR being 30.11% (totally 81 cases). ELISA identified 91 positive cases out of 263 specimens, with a detection rate of 34.60%, which was not significantly different from that by Nest-PCR. Immunocytochemistry detection results displayed that the polypeptide antibody could specifically reveal cervical exfoliated cells infected with HPV. Conclusion The HPV L1 polypeptide antibody can satisfactorily detect the HPV infection in cervical exfoliated cells, indicating that HPV L1 polypeptide antibody has great potential in developing HPV detection kit for cervical cancer preventive screening.