抗HPV16L1IgY抗体的制备及活性检测

目的制备出高特异性的抗HPV16L1IgY抗体,为HPV16L1蛋白的检测提供一种方法。方法用纯化HPV16L1蛋白免疫母鸡,分别收集鸡蛋,4℃下保存备用;经聚乙二醇法提取鸡蛋黄中IgY抗体;用酶联免疫吸附法检测IgY抗体效价的测定;采用免疫组织化学法通过对转染pcDNAEGFP-HPV16L1(含EGFP-HPV16L1融合基因)质粒的CHO细胞中L1蛋白的检测评价IgY抗体的特异性。结果3次免疫之后,IgY抗体效价达到1:10240,同时可特异性与转染pcDNAEGFP-HPV16L1(含EGFP-HPV16L1融合基因)质粒的CHO细胞中EGFP-HPV16L1结合。结论采用HPV16L1蛋白免疫母鸡,成功获得了较高效价的特异性IgY抗体,并可用于HPV16L1蛋白的细胞学检测,为IgY抗体在免疫组织化学检中的应用提供实验依据。

Transforming Activity of a Novel Mutant of HPV16 E6E7 Fusion Gene

An optimized recombinant HPV16 E6E7 fusion gene (HPV16 ofE6E7) was constructed according to codon usage for mammalian cell expression, and a mutant of HPV16 ofE6E7 fusion gene (HPV16 omfE6E7) was generated by site-directed mutagenesis at L57G, C113R for the E6 protein and C24G, E26G for the E7 protein for HPV16 ofE6E7 [patent pending (CN 101100672)]. The HPV16 omfE6E7 gene constructed in this work not only lost the transformation capability to NIH 3T3 cells and tumorigenicity in SCID mice, but also maintained very good stability and antigenicity. These results suggests that the HPV16 omfE6E7 gene should undergo further study for application as a safe antigen-specific therapeutic vaccine for HPV16-associated tumors.

Cisplatin sensitivity and mechanisms of anti-HPV16 E6-ribozyme on cervical carcinoma CaSKi cell line

Objective： The aim of this study was to study the cisplatin sensitizing effect and mechanism of anti-HPV16 E6- ribozyme on cervical carcinoma cell line. Methods： The anti-HPV16E6-ribozyme and empty eucaryotic expressing plasmids were transfected into CaSKi cell, which named as CaSKi-R, CaSKi-P respectively. E6 mRNA, the sensitivity to cisplatin, apoptosis rates, expression of p53, Bcl-2, Bax and C-myc proteins and mRNA were examined by Northem blot, MTT colorimetric assay, PI/Annexin V stained methods, flow cytometry anslysis and RT-PCR, respectively. Results： E6 mRNA was less in CaSKi-R than in CaSKi. The sensitivity of CaSKi-R cells to cisplatin was 2.28 and 2.21 times than that of CaSKi and CaSKi-P cells. The apoptotic rates in CaSKi, CaSKi-P and CaSKi-R cells was （18.9 ± 3.5）%, （19.7 ± 4.8）% and （40.4 ± 4.5）%. The apoptotic rates was increased in CaSKi-R than that of CaSKi cells treated with cisplatin （P = 0.003）. Comapred with CaSKi cell, the expression of p53 （P = 0.000）, Bax protein （P = 0.002） was significantly higher and the expression of Bcl-2 protein （P = 0.005）, C-myc protein （P = 0.005） was significantly lower in CaSKi-R than that of CaSKi cell treated with cisplatin. Comapred with CaSKi cell, the expression of p53, Bax mRNA in CaSKi-R cell treated with cisplatin increased, while Bcl-2, C-myc mRNA decreased. Conclusion： CaSKi-R cells transfected by anti-HPVE6-ribozyme increased the sensitivity to cisplatin. The increase of sensitivity to cisplatin in CaSKi-R cells may be associated with increasing expression of p53, Bax protein, and decreasing expression of C-myc, Bcl-2 proteins.

Correlation between Load of HPV 16 DNA in Cervical Cancer and HPV 16 DNA in Lymph Nodes

OBJECTIVE To determine the association between viral loadof human papillomavirus 16 (HPV16) DNA in the primary focusof cervical carcinoma and HPV16 DNA in pelvic lymph nodes.METHODS The HPV16 DNA load was measured by fluorescentquantitation polymerase chain reaction (FQ-PCR) in 17 primaryfoci. HPV16 DNA was detected by polymerase chain reaction(PCR) using HPV16 type-specific primers in 296 pelvic lymphnodes which were from 17 cases of cervical cancer.RESULTS The viral load of HPV16 DNA showed statisticallysignificant differences between tumors with a diameter of < 4cm and ≥ 4 cm (P < 0.05). Seven of 17 cervical cancer cases hadHPV16 DNA positive lymph nodes, designated as the positivegroup, while the remaining 10 without positive lymph nodes wasdesignated the negative group. The average load of HPV16 DNAshowed no significant difference between the 2 groups (P > 0.05).The load of HPV16 in the primary lesion was not associated withthat in the lymph nodes. There were 38 HPV16 DNA positivenodes in the total 296 nodes. The rate of positivity of HPV16 DNAin lymph nodes showed statistically significant differences inconsideration of maximum tumor diameter, tumor differentiation,histologic type, depth of myometial infiltration and the metastaticstatus of the nodes, respectively (P < 0.05).CONCLUSION Viral load of HPV16 in the primary cancer focuscorrelated with the quantity of tumor cells in the primary focusbut not with the existence of HPV DNA positive lymph nodes.Detection of HPV DNA may help to find the early metastases thatcannot be evaluated histopathologically, but the prognostic valueof HPV positive lymph nodes needs further examination.

Polymorphism in the upstream regulatory region of human papilloma virus type 16 from the cervical cancer biopsies in Xinjiang Uygur women

To investigate the mutations in the upstream regulatory region (URR) of human papillomavirus type 16 (HPV-16) from the cervical cancer biopsies in Xinjiang Uygur women and its relationship to the high incidence of cervical cancer in the southern Xinjiang, the tissue DNA was extracted from the cervical cancer biopsies, and the URR segment of HPV-16 DNA was amplified, sequenced and analyzed. Thereafter, the polymorphism of URR in HPV-16 was then analyzed. It was demonstrated that the positive rate detected for the presence of URR in HPV-16 was 89.47% (17/19). Compared with the previously published sequence in URR of prototype HPV-16, some mutations were detected in the sequence of URR. The mutations in 17 URR fragments of HPV-16 could be divided into 11 patterns (XJU-1 to XJU-11) at nucleic acid level, in which each of XJU-1 and XJU-4 accounted for 23.53% (4/17), and other patterns of mutation accounted for 5.88% (1/17) . In comparison with the URR of prototype HPV-16, the DNA identity of these patterns was 98.50%-99.68% . In these 17 URR fragments, two point mutations occurred at position 7192 (G to T) and position 7520 (G to A) and they appeared to be constant in Xinjiang area. These two mutations were ubiquitous in the Asia-American type and conferred strong infection activity and carcinogenicity of this virus. In addition, the mutations at position 7729 (A to C), position 7843 (A to G) and position 7792 (C to T) could enhance its transcription activity considerably. It is concluded that some mutations occur in URR gene of HPV-16 in the cervical cancer biopsies taken from Uygur women in Xinjiang area, suggesting that certain relationship exists among the mutations in URR of HPV-16, the phylogeny of HPV-16 and the high incidence of cervical cancer in southern part of Xinjiang area.

Construction of non-replicating recombinant vaccinia virus expressing HPV16 L1,L2E7 proteins

Objective： To construct a non-replicating vaccinia virus expressing human papillomavirus 16 （HPV16） L1, L2E7 proteins as a candidate vaccine for cervical cancer. Methods： Using vaccinia virus vector, we generated a strain of non-replicating recombinant vaccinia virus vaccine expressing HPV16 L1, L2E7 proteins by homologous recombination and identified by PCR and Westernloting. Results： We demonstrated that the L1, L2E7 gene of HPV16 were integrated into vaccinia genosome and could express L1, L2E7 protein stably when infected the CEF using PCR and Western-blot assay. Conclusion： NTVJL1/L2E7 can express L1, L2E7 protein of HPV16 and can be taken as a candidate vaccine for HPV16-associated diseases.

Transient Expression of Human Papillomavirus Type 16(HPV 16) mRNA in Normal Human Keratinocytes Transfected by pSV2-neo/16

Objective: To investigate the expression of HPV16 mRNA innormal human keratinocytes transfected with pSV2-neo/16. Methods: First human keratinocytes were cultured in theserum-free medium M154.Second, the plasmid pSV2-neo/16was transfected into the human keratinocytes using atransfecting reagent. Third, RT-PCR and Southern Blottingwere used to detect the expression of HPV16 mRNA and DNAin the transfected keratinocytes, respectively. Results: The expression of HPV 16 mRNA was successfullyamplified and an 110bp was detected by RT-PCR. A 7.9kbfragment was confirmed in the transfected keratinocytes bySouthern Blot analysis. Conclusion: HPV 16 mRNA and DNA were successfullydetected in the human keratinocytes.

Down-Modulation of Notch1 Expression in Cervical Cancer Is Associated with HPV-Induced Carcinogenesis

OBJECTIVE Notch1 signaling has been implicated intumorigenesis. The purpose of this study was to investigate theputative role of the Notch1 receptor in carcinogenesis and in theprogression of the cervical cancer. Since human papillomavirus(HPV) is a causative agent in cervical carcinoma, the interactionbetween Notch1 and HPV infection was examined.METHODS Forty cervical cancer samples and 30 normalcervical tissue specimens were examined using Western blot andRT-PCR to detect Notch1 protein and mRNA levels. HPV16 DNAwas examined in all samples using PCR.RESULTS The level of Notch1 protein expression wassignificantly lower in cervical cancer tissue than in normal tissue.Levels of Notch1 mRNA were found to be substantially downregulatedin cancer tissue. Notch1 protein expression levelswere significantly higher in carcinomas without HPV DNAthan that in carcinomas with HPV infection (55.5% vs. 3.3%, P <0.05). Down-modulation of Notch1 mRNA levels in carcinomawas demonstrated to be associated with HPVE6 transcription.Moreover, levels of Notch1 expression were shown to besignificantly higher in early stage disease than in advanced stagedisease (P = 0.001).CONCLUSION Down-modulation of Notch1 expressionprobably plays an important role in the late stages of HPVinducedcervical cancer.

pcDNAL1 genetic immunization can induce specific cell-mediated immune responses in C57BL/6 mice

OBJECTIVE: To investigate the specific cell-mediated immune efficacy of the an HPV16 prophylactic vaccine. METHODS: C57BL/6 mice were randomly divided into 3 groups: experimental group I (treated with pcDNA L1), control group II (treated with pcDNA3.1) and control group III (treated with PBS buffer). The mice were immunized three times during a three-week interval. Ten to fourteen days after the third inoculation, a footpad swelling test was used to detect delayed-type hypersensitivity (DTH) responses. Antigen-specific splenocyte proliferation assay and quantitation of IFN-gamma cells in splenocytes were performed by FACS assay. RESULTS: In the experimental group, splenocytes actively proliferated after stimulation with HPV16 VLP, and had developed a markedly larger amount of CD8(+) IFN-gamma(+) cells, which is an index for special CTL. Also, the footpad was significantly thickened upon inoculation with HPV16 VLP. CONCLUSION: Naked DNA vaccine of HPV16 L1 can induce specific cell-mediated immune responses in mice, which should be considered for evaluation of HPV16 DNA vaccine feasibility.