Fig 1.
Construction and identification of the DNA vaccine plasmid pEGFP-N1-Catsper1.
(A) The amplification of the whole Catsper1 ORF by recombinant PCR. The sequences marked with underline are the restriction enzyme sites of Xho I and BamH I. (B) Schematic diagram of pEGFP-N1 plasmid. EGFP was used as a report gene, and Kanr/Neor was a kanamycin resistance gene and used for colony screening of E.coli that transformed with plasmid. (C) Agarose gel electrophoresis of the recombinant PCR products. PCR products are, in order, the former half (Segment 1) of Catsper1 ORF with restriction enzyme site (1107 bp), the latter half (Segment 2) of Catsper1 ORF with restriction enzyme site (1106 bp) and the whole Catsper1 ORF with restriction enzyme sites (2079 bp). (D) Agarose gel electrophoresis of restriction enzyme digestion products of recombinant plasmid pEGFP-N1-Catsper1.
Fig 2.
In vitro expression of pEGFP-N1-Catsper1 in mouse N2a cells.
(A) mRNA expression detected by RT-PCR. Templates were cDNA from untreated mouse N2a cells, N2a cells transfected with pEGFP-N1-Catsper1 and mouse testis (positive control) respectively. (B) Expression of EGFP-CATSPER1 fusion protein in N2a cells transfected with pEGFP-N1-Catsper1. Scale bar = 100 μm.
Fig 3.
In vivo expression of pEGFP-N1-Catsper1 in mouse muscle tissue.
(A) Catsper1 mRNA expression detected by RT-PCR. Lane PBS: muscle tissue of PBS-injected mice; Lane 12 h to Lane 48h: muscle tissue of pEGFP-N1-Catsper1-injected mice 12, 24 and 48 hours after first injection respectively; Lane testis: mouse testis (positive control). (B) In vivo expression of EGFP-CATSPER1 fusion protein in the mouse muscle tissue. Scale bar = 20 μm.
Fig 4.
Antibody levels and specificity of pEGFP-N1-Catsper1-injected male mice.
(A) Serum IgG antibody response of immunized male mice by ELISA. M1-M6 represent 6 individual mice. The 0 week on horizontal axis represents the time point before the first immunization, and others represent the weeks after first immunization. P34 and Pp6 were used as coating antigen in the ELISA assay respectively. (B) The specificity of antibodies in serum of immunized mice. Scale bar = 20 μm.
Table 1.
Fertility test of immunized male mice.
Fig 5.
Changes in sperm motility and histology of testis and epididymis.
(A) Sperm motility and hyperactivated motility of immunized mice. **P<0.001. (B) Histology of testis and epididymis of immunized mice. Scale bar of testis = 50 μm, scale bar of epididymis = 20 μm.