| Blackground:The human population is projected to exceed a phenomenal10billion by the year2050. Major increase in human population will take place in developing countries as compare to developed countries. These information indicate the burning need for new contraceptive methods. Immunocontraception has been considered as a promising approach for a long time with several potential advantages. One candidate antigen of epididymal proteins is cysteine-rich secretory protein-1(CRISP-1), an androgen-dependent protein that associates with the sperm surface during epididymal transit and participates in gamete fusion throμgh complementary sites localized on the egg surface. Immunization with purified CRISP-1from native source or obtained by recombinant DNA technology has been shown to elicit a specific antibody response and significantly reduce the fertility in several animal models throμgh a mechanism involving the entry of the antibodies to the reproductive tract and their specific interference with the sperm fertilizing ability, postulating that the CRISP-1protein might be a putative target for post-testicular male contraception. Another alternate possibility of generating an immune response against sperm proteins, in vivo, is to directly administer the sperm proteins to a host in which plasmid DNA encodes either individual or a collection of antigens. However, as DNA always carries negative discharge, it is hard to pass throμgh cell membrane and easy to be degenerated by nuclease in vivo. Chitosan(CS), a positively charged natural polymers, with good biocompatibility, biodegradability and non-toxicity, has been widely used in pharmaceutical industry as a new vector system in the recent years. Previous studies have confirmed that chitosan may play a synergistic role in optimizing the immune effect when it was administered with different antigen together. In this study, mouse Crisp-1cDNA was selected as the target genes, eukaryotic expression plasmid pcDNA3.1-Crisp-1was constructed using recombinant DNA technology, and chitosan-pcDNA3.1-Crisp-1nanocompositesits(CS/DNA NPs) was prepared using the complex coagulation technology. Finally, we compared the immune responses and security among CS/DNA vaccines, naked DNA and purified r-mCRISP-1protein, in order to lay a foundation for the development of a new male contraceptive vaccine. Objective:1. To construct the eukaryotic expression plsmid of mouse cysteine-rich secretory protein-1(Crisp-1).2. To construct chitosan nanoparticles containing Crisp-1DNA vaccine.3. To evaluate the immune contraceptive effects of cysteine-rich secretory protein-1(Crisp-1) DNA contraceptive vaccine on male and female BALB/c mice and the efficacy and security of CS NPs as a DNA contraceptive vaccine vector.Methods:1. The eukaryotic expression plasmid pcDNA3.1-Crisp-1was constructed using Gene recombination technology, and then the recombinant plasmid was digested with BamHI and HindⅢ and confirmed by DNA sequence analysis. COS-7cells were transiently transfected in vitro with either pcDNA vector alone or with pcDNA3.1-Crisp-1plasmid by Lipofectamine2000TM procedure. After48hours, the mCRISP-1mRNA expression profile in the mammalian cells was determined by reverse transcription-polymerase chain reaction (RT-PCR) and the localization of expressed mCRISP-1protein in COS-7cells was observed by indirect immunofluorescence (IF).2. Chitosan-pcDNA3.1-Crisp-1(CS/DNA) plasmid Nanoparticles (NPs) were prepared using complex coagulation technology at the DNA concentration of50μg/ml,100μg/ml,200μg/ml respectively. The loading efficiency of DNA (%) onto CS NPs was measured using a UV spectrophotometer at260nm. The morphology of the CS/DNA NPs was observed using transmission electron microscope (TEM) and the particle size and zeta potential were measured on a nanoparticle size analyzer. The binding of DNA with CS and the protective effect of NPs on DNA were determined by1%agarose gel electrophoresis. CS/DNA NPs was transfected into COS-7cells in vitro directly, the cytotoxicity on cell was evaluated by MTT compared to Lipofectamine2000TM. qRT-PCR and IF were used to determine the expression of mCrisp-1in COS-7cells.3. Male and female BALB/c were divided into5groups randomly:0.9%NS group, pcDNA3.1vector group, pcDNA3.1-Crisp-1group, CS/DNA NPs group, and r-mCRISP-1group. Animals were immunized with50μg dose of DNA vaccine or r-mCrisp-1in intramuscularly (i.m.) in the anterior tibialis muscle. Two booster injections were given at2and4weeks, respectively. Blood was collected from each animal once before the first injection and then2,4,6,8,10and12weeks after the first injection Antibody titers were determined by enzyme-linked immunosorbent assay (ELISA). Specificity of anti-mCRISP-1antibody was carried out by western blot and neutralization experiment. Two weeks after the third immunization, all the mice were mated with the normal contra-sexual fertile mice. The appearance of a vaginal copulation plμg the next morning indicated a successful mating. The females were allowed to deliver in the next3weeks in a separate cage and the number of pups was counted. The syndroms of allergic, inflammatory and toxicity on immunized mice were observed during the administration time. The vital organs and reproductive organs were collected from immunized mice8weeks after the last injection for pathological examination.Results1. The recombinant plasmid pcDNA3.1-CRISP-1was digested with BamHI and HindⅢ, two bands were obtained:The larger about5.4k was the fragment of pcDNA3.1vector and the smaller about750bp was the fragment of Crisp-1. The aimed segment was further sequenced and the DNA sequence analysis revealed the obtained Crisp-1cDNA gene sequence was identical to the sequence from genebank. The RT-PCR results showed that a specific band approximately735bp was detected in the pcDNA-mCRISP-1transfected cells, while no band was detected in neither pcDNA3.1vector transfected cells nor untreated normal cells. Indirect immunofluorescence revealed the presence of mCRISP-1in pcDNA-mCRISP-1transfected cells. However, COS-7cells transfected with pcDNA3.1vector alone and untreated normal cells failed to show any fluorescence.2. The UV spectrophotometer results show that the plasmid embedding rate are all more than90%at the three concentrations. However, the highest encapsulation efficiency was observed at the concentration of100μg/mL (94.09±0.17%), which was significantly higher than the other two groups (p＜0.05). The imaging by TEM demonstrates that the shape of CS/DNA NPs is spherical or elliptical, and the size of those particles was optimized to150-200nm with a narrow distribution. The nano-particle analyzer results show that the mean diameter of CS/DNA NPs is189.3nm, and the polydispersity index is0.459with a narrow particle size distribution range. The zeta potential is approximately+0.2mV. The binding capacity of CS with DNA was evaluated using1%agarose gel electrophoresis and the results show that the migration of plasmid DNA was completely retarded in the sample wells. In contrast to the control naked DNA, plasmid DNA in the CS/DNA NPs was protected from DNase I. In MTT assay, the cell viability for naked DNA and for the CS/DNA NPs had no significantly difference (p>0.05), however, that for Lipofectamine2000Tm was significantly reduced (p<0.01). Indirect immunofluorescence revealed the bright green fluorescence can be observed both in CS/DNA NPs transfected cells and Lipofectamine2000TM ransfected cells. However, COS-7cells transfected with pcDNA3.1vector alone or untreated normal cells failed to show any fluorescence. Real-time PCR analysis showed that there was no Crisp-1mRNA expressed in neither untreated cells nor pcDNA3.1vector transfected cells. The Crisp-1mRNA expression level of Lipofectamine2000TM transfected cells slightly higher than that of CS/DNA NPs transfected cells, however, there was not significant difference (p>0.05).3. ELISA results showed that there was no anti-mCRISP-1specific antibodies induced by the pcDNA3.1vector and0.9%NS, but pcDNA3.1-Crisp-1immunized male and female mice produced specific antibodies just2weeks after the first inoculation, which was significantly higher than the pcDNA3.1vector and0.9%NS groups (p＜0.05). The highest antibody titers were observed2weeks after the last injection and decreased slowly8weeks after the third boost. The titers of anti-mCRISP-1antibodies from CS/DNA NPs and r-mCrisp-1protein immunized mice were significantly higher than that of pcDNA3.1-Crisp-1immunized mice (p＜0.05). However, there was no significant difference between the CS/DNA NPs immunized mice and pcDNA3.1-Crisp-1immunized mice (p＞0.05). The immunoreactivity of the antisera generated in immunized mice was analyzed by Western blotting. The anti-mCRISP-1antibodies generated by pcDNA3.1-Crisp-1, CS/DNA NPs and r-mCRISP-1protein immunized mice detected a faint band of～36kDa and a strong band of～32kDa on purified r-mCRISP-1and a a single band of～30kDa on sperm extracts. No reactivity was observed with pooled pre-immune serum. In addition, neither visible bands appeared in post-immune sera from the DNA vector nor0.9%NS immunized mice. Neutralization experiment results showed that the immunoreactivity with native CRISP-1in sperm extracts and r-mCRISP-1protein were completely inhibited in the presence of r-mCRISP-1protein. The mating test results showed that the fertility rates and mean litter size of female mice in each group were:0.9%NS (100%,10.75±0.55), pcDNA3.1vector (90.9%,10.69±0.44), pcDN A3.1-Crisp-1(45.5%,6.80±0.58), CS/DNA NPs (25%,4.00±0.57), r-mCRISP-1protein(25%,4.33±0.33). The fertility rates and mean litter size of male mice in each group were:0.9%NS (100%,10.57±0.56), pcDNA3.1vector (100%,10.43±0.55), CS/DNA NPs (26.7%,3.75±0.48), r-mCRISP-1protein (26.7%,3.75±0.25). In comparison with0.9%NS group, the fertility rates and litter size of the both male and female animals immunized with pcDNA3.1vector have not significant reduction, however, those of the other three groups showed different degrees reduction. The reduction in the fertility rates and litter size of pcDNA3.1-mCRISP-1immunized mice were significantly higher than that of0.9%NS and pcDNA3.1vector immunized mice, however, they were significantly lower than that of CS/DNA NPs and r-mCRISP-1protein immunized mice. There was no significant difference in the fertility rates and litter size between CS/DNA NPs immunized mice and r-mCRISP-1protein immunized mice (p＞0.05). There was no significant difference in the fertility rates and litter size between male and female mice, too (p＞0.05). During the period of administration, the mice in each group were undetected obvious exception. In comparison with the control group, the tissues from experimental groups showed normal structure without signs of inflammatory infiltration.ConclusionIn this study, we successfully constructed the eukaryotic expression plasmid pcDN A3.1-Crisp-1and CS/DNA NPs. In spite of the pcDNA3.1-Crisp-1DNA vaccine can reduce the mice fertility, the contraceptive effect is not ideal. CS NPs can improve the pcDNA3.1-Crisp-1DNA vaccine immune response, and has good security. |
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