修饰猪IGF-1基因壳聚糖纳米颗粒对小鼠生长及免疫的促进效应(英文)

Improvement of Growth and Immunity of Mice by Truncated Porcine IGF-1Gene Encapsulated in Chitosan Nanoparticles

目的研究比较修饰后IGF-1基因对小鼠免疫机能及生长的影响,探索研制调控动物免疫和生长发育的新型安全高效的分子制剂。方法通过重叠PCR扩增技术克隆得到新型短链IGF-1基因,将其分别构建到原核表达载体pGEX-4T-1及真核表达载体VR1020中,得到重组质粒pGG,pGF,VRG和VRF,分别进行体外实验检测器表达活性;然后用壳聚糖和mPEG-PEI修饰得壳聚糖通过离子交联包裹重组质粒,将包裹后形成的纳米颗粒,按0.1mg质粒/只的剂量接种3周龄昆明小鼠。在免疫前及免疫后每隔一周采血,检测小鼠体重变化,血常规检测免疫细胞数量的变化,流式细胞分型技术检测CD4+/CD8+标记的T细胞,并且通过MTT实验检测上清对L-02细胞的增殖作用。结果发现免疫后14～42d之间,与对照组相比实验组VRI、VRG小鼠外周血中淋巴细胞、CD4+T细胞量明显升高(P<0.05);并且免疫后14～56d之间,与对照组相比,实验组VRI、VRG对小鼠生长的促进作用也非常显著(P<0.05);另外mPEG-PEI-CS和CS包裹VRI、VRG接种的实验组小鼠血清对L-02细胞具有明显的增殖作用。结论我们的研究首次证明在小鼠免疫后14～42d中,VRG/F与对照组相比,对L-02细胞具有明显的增殖作用(P<0.05)。这些结果说明了短链的IGF-1基因能够促进接种后小鼠的生长发育,提高其免疫功能,为进一步研究控制动物传染疾病安全高效的生物制剂奠定了基础。

Objective To compare of effect of porcine IGF-1 and modified IGF-1 gene on the growth and immunity of mice, and develop safe and effective molecular regulator for immunity and growth of animal. Methods The novel truncated porcine IGF-1 genes were cloned with overlapping PCR extension technique, and then respectively subcloned into prokaryot- ic and eukaryotic expression plasmid, pGEX-4T-1 and VR1020, respectively designed as recombinant pGG, pGF, VRG and VRF. They were respectively expressed in vitro to evaluate their bioactivity, and packed with ehitosan （CS） and mPEG-PEI derived CS prepared by ionotropic gelation method. They were then utilized to inoculate 3-week-old Kunming mice intramuscularly at the dose of 0.1 mg/per mouse, respectively. The blood were collected from the mice before and af- ter inoculation on 0, 2, 4, 6, 7 and 8 weeks to detect the changes of weight, immune cells, CD4^＋/CD8^ ＋ T cells by FCM and influence on L-02 cell proliferation by MTT. Results It was found that the lymphocytes significantly increased in the blood of VRI and VRG groups from 14 to 42 clays post inoculation compared with that of control mice, and the number of CD4^＋ T cells was also remarkably higher than the control （ P 〈 0.05 ）. The growth performance of VRI and VRG groups were obviously better than the control group from the 14^th day to 56^th day after inoculation （ P 〈 0.05 ）. The sera from mPEG-PEI-CS trapped VRI/VRG and CS-VRI/VRG could significantly promote the proliferation of L-02 cells. Conclusion Our results firstly proved that VRG/F could markedly accelerate proliferation of L-02 cell from 14 to 42 days after inocula- tion in comparison with that of the control （ P 〈 0.05 ）. These suggested that the growth and immunity of inoculated mice could be promoted by the truncated IGF-1 gene, which would inspire the development of safe and effective adjuvant to con- trol of infectious diseases of animal in the future.