[Prokaryotic expression, purification of human papillomavirus type 16 E1 protein and preparation of it's antiserum].

AIM: To express and purify the human papillomavirus type 16 E1 protein in E.coli and prepare the antibody against HPV-16 E1. METHODS: HPV-16 E1 gene was amplified by PCR and cloned into prokaryotic expression vector pMAL-p2x, and the recombinant plasmid was transformed into E.coil BL-21. We optimize the soluble expression condition of fusion protein by induction with different IPTG concentration and different temperature. The expressed fusion protein was purified by mahose affinity column Chromatography. To prepare the anti-serum, New Zealand white rabbits were immunized with purified HPV-16 E1 protein via hypodermic and volar. Western blot and ELISA analyzed the serum's specificity against HPV-16 E1 and serum titers. RESULTS: Restriction endonuclease analysis and DNA sequencing showed HPV-16 E1 was cloned into the plasmid pMAL-p2x. Based on the optimization experiments, it concluded that the best soluble expression conditions for the HPV-16 E1 fusion protein involved addition of IPTG to a final concentration of 0.5 mmol/L and then further incubation at 28°C. The purity of the HPV-16 E1 fusion protein was over 95.7% after purification. ELISA and Western blotting showed the titers of the anti-serum were above 1:640 000, and the anti-serum can specifically bind with HPV-16 E1 protein. CONCLUSION: We have ingathered the HPV-16 E1 fusion protein by expressing in E.coli and purifying, and the antibody against HPV-16 E1 was prepared with the fusion protein immunizing New Zealand white rabbits. This work will provide an antigen and detection antibody for further study on the HPV-16 E1 function.

[Construction of codon-optimized HPV16 capsid genes in the eukaryotic co-expression vector and transfection of cells].

AIM: To construct the eukaryotic co-expression vector pcDNA3.1-L1-IRES-L2 for harvesting sufficient amounts of infectious HPV16. METHODS: The amplified codon-optimized HPV16 capsid genes from 988 plasmid by PCR were cloned into pCR-XL-TOPO vector and then subcloned into eukaryotic expressing vector pcDNA3.1(+). Thus, eukaryotic co-expression vector pcDNA3.1-L1-IRES-L2 capable of expressing HPV L1 gene and L2 gene was constructed. This eukaryotic vector was verified by enzymolysis and sequencing. The transcription of capsid genes was observed in vivo and in vitro by hydrodynamics-based transfection and liposome-mediated transfection. The expression of L1 gene in 293T cells was examined by Western blot. RESULTS: The eukaryotic co-expression vector pcDNA3.1-L1-IRES-L2 was successfully constructed and verified by enzymolysis and sequencing.The recombinant plasmid L1 and L2 genes were transcripted in the livers of the rats and 293T cells.Cytopathic effect (CPE) occurred after the transfected with pcDNA3.1-L1-IRES-L2 into 293T cells, indicating that the two capsid genes were expressed.Western blot detection showed the recombinant plasmid L1 protein was expressed in 293T cells. CONCLUSION: The pcDNA3h1-L1-IRES-L2 has been constructed successfully, which lays a foundation for in-dept study on the infectious mechanism of HPV16.

[Immunocontraceptive effect of DNA vaccines of Lagurus lagurus zona pellucida 3 fusioned with C-terminal of HSP70].

AIM: To investigate the immunocontraceptive effect of Lagurus lagurus zona pellucida 3 gene (LZP3) fused by Mycobacterium tuberculosis HSP70 and C-terminal of HSP70. METHODS: Two immunocontractive recombinant plasmids pcD-L-HSP70 and pcD-L-HSP70C were constructed using LZP3. Meanwhile, pcD-L and pcD-ACLC which were effective immunocontraceptive vaccines were studied as control. RESULTS: After the LZP3 genes were expressed in the livers of mice with the introduction of hydronamic transfection instead of traditional HeLa cell transfection by RT-PCR, NIH mice were immunized with ZP3 DNA vaccines. The results of ELISA showed that all of the recombinants in mice elicited specific anti-ZP3 antibodies which were combined with the ZP3 of oocytes from the immunized mice in immunofluorescence assay. MTS assay showed that the lymphocytes in all groups were proliferated by stimulating the recombinant LZP3 with no disruption of follicular in ovaries, especially those in groups of pcD-ACLC and pcD-L-HSP70C (P<0.01). Antifertility experiments showed that three groups (besides pcD-L-HSP70) enhanced the sterile effects (P<0.05), especially those in the groups of pcD-ACLC and pcD-L-HSP70C (P<0.01). CONCLUSION: LZP3 fused by the C-terminal of mycobacterium tuberculosis heat shock protein HSP70 had a good immunocontraceptive effect while LZP3 fused by Mycobacterium tuberculosis heat shock protein HSP70 had a poor effect on birth control. So the C-terminal of Mycobacterium tuberculosis heat shock protein HSP70 might be a better candidate to deliver the adjuvant function in LZP3 DNA immunocontraceptive vaccination than the complete HSP70 molecule.

[Construction and expression of a recombinant adenovirus with LZP3].

AIM: To explore a new immunocontraceptive vaccine and construct an attenuated recombinant adenoviral vaccine against Lagurus lagurus zona pellucida 3(LZP3). METHODS: LZP3 gene was subcloned into the shuttle vector pShuttle-CMV, and then a two-step transformation procedure was employed to construct a recombinant adenoviral plasmid with LZP3, which was digested with Pac I and transfected into HEK293 cells to package recombinant adenovirus particles. Finally, HeLa cells were infected by the recombinant adenovirus. LZP3 gene was detected from the recombinant virus by PCR, and its transcription and expression were analyzed by RT-PCR and Western blot. RESULTS: Recombinant adenovirus vector pAd-LZP3 with LZP3 gene was constructed by homologous recombination in E.coli, and a recombinant adenovirus was obtained by transfecting HEK293 cells with pAd-LZP3. PCR test indicated that LZP3 gene was successfully integrated into the adenoviral genome, and the titer of the recombinant adenovirus reached 1.2x10(10) pfu/L. The transcription and expression of LZP3 gene in the infected HeLa cells were confirmed by RT-PCR and Western blot. CONCLUSION: The recombinant adenovirus RAd-LZP3 can be successfully expressed in the infected HeLa cells, which lays the foundation for further researches into immunizing animals with RAd-LZP3.

[Prokaryotic expression of HPV 16 L1 gene and identification of its immune activity].

AIM: To express the major capsid protein of human papillomavirus type 16 L1(HPV 16 L1) in E.coli and identify its immune activity. METHODS: The L1 gene of HPV 16 was cloned into the expression vector pThioHisC. The recombinant expression vector was transformed into E.coli, and the HisC-L1 protein was expressed under IPTG induction. The fusion protein was characterized by SDS-PAGE and Western blot. RESULTS: The HPV16-L1 gene in plasmid pThioHisC was expressed in E.coli as a fusion protein with M(r) about 70,800. The fusion protein reacted specifically with antibodies against HPV16-L1. CONCLUSION: The HPV-16 L1 gene was expressed successfully in E.coli, which provides necessary basis for preparing HPV-16 L1 vaccine in human.

[Analysis of human papillomavirus 16 E6 oncogene mutation in Xinjiang Uygur women with cervical carcinoma].

BACKGROUND & OBJECTIVE: High-risk human papillomaviruses(HPVs),such as HPV16, and HPV18,are major causes of cervical cancer (CC), and HPV16 was found most frequently in CC patients. HPV16E6 is one of major oncogenes. In some region, specific E6 mutation is considered as dangerous factor causing CC. There is a very high incidence of CC in southern Xinjiang, where the Uygur are the majority. As we reported before, we found HPV16E6 mutation from this district. This study was designed to investigate distribution of the mutation in CC of Xinjiang Uygur women, and the relationship between the mutation and high incidence of CC in southern Xinjiang. METHODS: The tissue DNA was extracted from 35 CC biopsies of Xinjiang Uygur Women. HPV16E6 gene was amplified by polymerase chain reaction (PCR) from the CC tissue DNA. The PCR fragments were sequenced and analyzed. RESULTS: The result of PCR showed that the positive rate of HPV16E6 was 82.86%(29/35); 26 of these 29 PCR fragments were sequenced and analyzed, 15 of them maintained prototype (57.69%), 11 have L83V mutation (34.62%), and 2 have L83V/D63E mutation (7.69%). CONCLUSIONS: There is mutation within the HPV16E6 gene in CC of Xinjiang Uygur women. Our research suggested that the distribution of HPV16 prototype and HPV16E6 mutation might be associated with high incidence of CC in southern Xinjiang.

[Mutations of human papillomavirus (HPV) 16 type L1 genes from cervical carcinoma biopsies in southern Xinjiang Uygur women].

OBJECTIVE: To study the mutations of Human Papillomavirus (HPV) 16 type L1 genes of cervical carcinoma biopsies from Uygur women in Southern Xinjiang, and analyze changes of L1 protein function. METHODS: The tissue DNA was extracted from cervical carcinoma biopsies. HPV16 L1 genes were amplified by PCR from the DNA HPV16 type L1 genes were sequenced and analyzed. RESULTS: The result of PCR showed that the positive rate of HPV16 L1 was 84.21% (16/19). These DNA were sequenced, and we found some mutations in comparison with the previously published sequence of prototype HPV16 L1. Some of the mutations changed the triplet codes, subsequently led to changes of amino acids. The mutations of all thirteen HPV16 L1 fragments formed six patterns (XJL1-1 approximately XJL1-6) at nucleic acid level. Compare to HPV16 prototype, their homology were 99.69% to 99.87%. There were four mutations in nucleic acid sequences of XJL1-1, which occurred also in XJL1-2 approximately XJL1-6. Moreover, there are other mutations in XJL1-2 approximately XJL1-6 besides the four mutations in XJ L1-1. The mutations of all thirteen HPV16 L1 fragments formed four patterns at amino acid level, among the mutations XJL1-1/2/3 was by 76.92% (8/13). CONCLUSION: HPV16 type L1 genes from cervical carcinoma biopsies occurred some mutations in Uygur women from southern Xinjiang, and formed several patterns as well as mainstream pattern. The mutations of L1 proteins changed its hydrophobicity and antigenicity. The research suggested that the mutations of HPV16 type L1 genes associated with HPV16 phylogenesis and escape from immune recognition.