Dan Xingang, Han Li, Riaz Hasan, Luo Xuan, Liu Xiaoran, Chong Zhenglu, Yang Liguo
Key Lab of Education Ministry of China in Agricultural Animal Genetics, Breeding and Reproduction, Huazhong Agricultural University, Wuhan 430070, P.R. China.
Exp Anim. 2016;65(1):17-25. doi: 10.1538/expanim.15-0044. Epub 2015 Oct 2.
To further improve fertility of animals, a novel gene RFRP-3 (RF-amide related peptide-3, RFRP-3) was used to construct DNA vaccines with INH α (1-32) (inhibin, INH) fragment for the first time. The aim of this study was to evaluate the effects of novel DNA vaccines on fertility in mice. Synthesized SINH and SRFRP (INH and RFRP genes were separately ligated to the C-terminus of the small envelope protein of the hepatitis B virus (HBV-S) gene) fragments were inserted into multiple cloning site of pIRES vector to develop p-SINH/SRFRP. The synthesized tissue plasminogen activator (TPA) signal sequence was then inserted into the p-SINH/SRFRP to construct p-TPA-SINH/TPA-SFRFP. Meanwhile, p-SINH was prepared and considered as positive control. Forty Kunming mice were equally divided into four groups and respectively immunized by electroporation with p-SINH, p-SINH/SRFRP and p-TPA-SINH/TPA-SRFRP vaccine (three times at 2 weeks interval) and saline as control. Results showed that the average antibodies (P/N value) of anti-INH and anti-RFRP in mice inoculated with p-TPA-SINH/TPA-SFRFP were significantly higher (P<0.05) than those inoculated with p-SINH/SRFRP and the positive rates were 100% (anti-INH) and 90% (anti-RFRP) respectively, at 2 weeks after the third immunization. Litter size of mice immunized with the three recombinant plasmids was higher (P<0.05) than that of the control, and litter size of mice immunized with p-TPA-SINH/TPA-SRFRP significantly increased (P<0.05) compared with p-SINH. These results suggested that the p-TPA-SINH/TPA-SRFRP harboring INH and RFRP genes was successfully constructed and had good immunogenicity, and might effectively increase litter size.
为进一步提高动物的繁殖力，首次使用一种新型基因RFRP - 3（RF - 酰胺相关肽 - 3，RFRP - 3）与抑制素α（1 - 32）（抑制素，INH）片段构建DNA疫苗。本研究的目的是评估新型DNA疫苗对小鼠繁殖力的影响。将合成的SINH和SRFRP（INH和RFRP基因分别连接到乙肝病毒小表面蛋白（HBV - S）基因的C末端）片段插入pIRES载体的多克隆位点，构建p - SINH/SRFRP。然后将合成的组织纤溶酶原激活剂（TPA）信号序列插入p - SINH/SRFRP中，构建p - TPA - SINH/TPA - SFRFP。同时，制备p - SINH作为阳性对照。40只昆明小鼠平均分为四组，分别通过电穿孔用p - SINH、p - SINH/SRFRP和p - TPA - SINH/TPA - SRFRP疫苗（间隔2周接种3次）进行免疫，以生理盐水作为对照。结果显示，在第三次免疫后2周，接种p - TPA - SINH/TPA - SFRFP的小鼠中抗INH和抗RFRP的平均抗体（P/N值）显著高于接种p - SINH/SRFRP的小鼠（P<0.05），抗INH的阳性率为100%，抗RFRP的阳性率为90%。用三种重组质粒免疫的小鼠产仔数高于对照组（P<0.05），与p - SINH相比，用p - TPA - SINH/TPA - SRFRP免疫的小鼠产仔数显著增加（P<0.05）。这些结果表明，携带INH和RFRP基因的p - TPA - SINH/TPA - SRFRP成功构建且具有良好的免疫原性，可能有效增加产仔数。
