| Objective: Human papillomaviruses (HPVs) infect people's epithelial cells in the skin and mucosa. They are closely linked with the benign and malignant hyperplasia of epithelial cells. Some subsets of HPVs cause benign hyperplasia of epithelial cells such as skin warts, condyloma acuminata and so on. The other subsets also cause genital cancers, such as cervical cancer, and cancers of skin and the oral cavity. Due to no specific therapeutic methods, HPV infection is highly detrimental to human's health. Therefore, there is a great need to develop a safe and effective vaccine to prevent and treat papillomavirus-induced diseases. Since we are not able to cultivate HPV in large quantities in the vitro at present, it is difficult to develop a traditional vaccine. Genetic immunization with naked DNA has emerged as an important strategy for vaccine development. DNA vaccines have a lot of advantages. For example, they are easy to prepare, safe and stable and capable of inducing persistent immune responses. The E7 oncoprotein of HPV16 is potentially an ideal target of immune therapy. Study showed that the HPV16 E7 vaccines can induce persistent cell-mediated immunity and humoral immunity, protect the mice from the attacks of HPV16 positive cancer cells, suppress the growth of the existing tumor or even clear it. However, when the vaccine is used on primates, its immunogenicity decreases, and when it is used on humans, the immune responses it induces are even weaker. As the vaccine will ultimately be used on humans, its immunogenicity must be enhanced. Research shows that immune response can be enhanced by co-administering some cytokines. Interleukin-15 (IL-15) is a recently identified cytokine which shares many biological properties of interleukin-2 (IL-2) such as activation of T cell, particularly CD8+CTLs. Unlike IL-2, it also plays a central role in the development and function of NK cells. IL-15 has been used in some experiments as an adjuvant and has significantly enhanced the induction of Ag-specific immune responses. But so far, there have been no reports concerning its effects on the immunity of HPV16 E7. This experiment subcloned IL-15 and constructed its eucaryon plasmids, which were injected along with the HPV16 E7 vaccine into BALB/c mice. Then, we investigated its effects on the Ag-specific immune responses of the mice.Methods: The pGEM-T-IL-15 was digested with EcoRI and BamHI. IL-15 gene fragments which had 500bp were withdrawn by Sliver Beads gel Extract, IL-15 gene fragment and plasmid pcDNA3.1 were cut with the same enzymes and linked to construct a recombinant eukaryotic expression plasmid pcDNA3.1-IL-15. E.coli DH5α were transformed in Ampicillin LB culture and the recombinant clones were selected. PcDNA3.1-IL-15 DNA was identified by digested with EcoRI and BamHI. Then it was prepared and purified. pcDNA3.1-E7,pcDNA3.1 was transformed E.coli DH5α. Positive clones were selected, identified by digested, prepared and purified in the same way. All the plasmids are diluted to the concentration needed and are used for immunizing mice. Female BALB/c mice, which were divided randomly into six groups, were immunized at the 6th week of age. All animals were pretreated with 100ul of 25% cane sugar each in tibialis anterio muscle 30min before the DNA immunization. One group of mice were then injected with a mixture of pcDNA3.1-IL-15 and pcDNA3.1-E7, 100ug per mice, boostered with the same DNA vaccine after two weeks for three times. Mice were immunized respectively with mixture of pcDNA3.1 and pcDNA3.1-E7,pcDNA3.1-E7,pcDNA3.1-IL-15,pcDNA3.1 and N.S. with the same method as control groups. After the last immunization, mice were killed, and the concentration of serum IFN-γ were determined by specific enzyme-linked immunosorbent assay (ELISA). Single-cell suspensions of splenocytes were prepared from mice of every group, and were restimulated with HPV16 E7 protein or mediam only. Then, the T cell proliferation assay was measured by the MTT method. The disparity vale of OD570 represents the d...
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