背景：1型糖尿病（T1D）是一种因自身效应性T细胞攻击胰岛β细胞引发的自身免疫性疾病。T1D的传统治疗依赖于长期注射胰岛素，而新上市的Afrezza、Teplizumab等新药物的高昂成本限制了其广泛应用，且无法逆转疾病进程。基于口服耐受的作用机理，使得机体产生T1D自身抗原耐受是预防和治疗T1D的最好方式。然而，口服抗原面临在胃肠环境中被降解、递送效率低等挑战。利用乳酸菌这类肠道益生菌通过胞质表达抗原可保护抗原完整性，同时融合肠道M细胞靶向肽CKS9可增强抗原向肠道派氏结的递送，促进抗原提呈细胞摄取。乳酸菌载体与肠道M细胞靶向技术的结合为T1D免疫耐受治疗开辟新路径，优化的递送系统实现了T1D口服疫苗的安全性、稳定性、高效性，有望实现T1D的早期干预与根治。

方法：本研究利用乳酸球菌表达系统，将肠道M细胞靶向肽CKS9与截选的GAD65191-320及INS17-110通过G4S连接序列连接并构建到pNZ8148质粒上并转入NZ9000乳酸球菌中，随后进行考马斯亮蓝染色以及Western blot实验确定融合蛋白质的表达。在动物实验中，上述菌种以1010 CFU/mL (100 μL)连续7天灌胃免疫，间隔两周后重复免疫步骤以完成口服免疫程序，对重组乳酸球菌在小鼠肠道内定植、肠道M细胞靶向性以及疫苗免疫原性进行鉴定，随后进疫苗对STZ诱导的T1D模型BALB/c小鼠以及非肥胖型糖尿病（NOD）小鼠的保护性评价。

结果：1.设计的CGIH、HGIC、GIH、SGIH重组乳酸球菌疫苗均构建成功，且100μL剂量的口服浓度达到109CFU/mL即可产生较好的T1D耐受诱导作用；2.携带C序列及S序列的乳酸球菌于小鼠肠道定植水平是未携带上述序列乳酸球菌定植水平的数十倍（其中于BALB/c小鼠中提升近40倍，NOD小鼠中提升近60倍）；3.口服重组乳酸球菌靶向疫苗能够建立T1D免疫耐受生境，延缓T1D的发病进程，且C序列位于GI序列后的HGIC靶向疫苗具有更好作用；4. anti-CD25单克隆抗体清除Treg细胞后，重组乳酸球菌靶向疫苗能够刺激Treg细胞增殖分化，恢复Treg细胞耗竭所引发的病理损伤。

结论：成功构建出四种能够表达具有免疫原性的GAD65191-320 + INS17-110融合蛋白质的重组乳酸球菌疫苗。CGIH、HGIC重组乳酸球菌靶向疫苗具有肠道M细胞靶向性，且在链脲佐菌素（STZ）诱导的T1D模型BALB/c小鼠中，NOD小鼠中均展现出了较好的保护作用。本研究将为T1D自身免疫性疾病的治疗及疫苗研发带来一定的理论参考。

Background: Type 1 diabetes (T1D) is an autoimmune disease caused by autoreactive effector T cells attacking pancreatic β cells. Traditional T1D treatment relies on long-term insulin injections, while newly approved drugs such as Afrezza and Teplizumab are limited by high costs and fail to reverse disease progression. Inducing immune tolerance to T1D autoantigens through oral tolerance mechanisms represents an optimal strategy for T1D prevention and treatment. However, oral antigens face challenges such as degradation in the gastrointestinal tract and low delivery efficiency. Utilizing Lactococcus lactis (L. lactis) as carriers to cytoplasmically express antigens can protect antigen integrity, while fusion with the M cell-targeting peptide CKS9 enhances antigen delivery to intestinal Peyer’s patches, promoting uptake by antigen-presenting cells. The combination of L. lactis carriers and M cell-targeting technology opens new avenues for T1D immune tolerance therapy. This optimized delivery system ensures safety, stability, and efficacy of oral T1D vaccines, offering potential for early intervention and disease cure. Methods: This study utilized the L. lactis expression system to construct a fusion protein by linking the intestinal M cell-targeting peptide CKS9 with truncated GAD65191-320 and INS17-110 through a G4S linker sequence. The constructed gene was cloned into the pNZ8148 plasmid and transformed into L. lactis NZ9000. Protein expression was confirmed through Coomassie blue staining and Western blot analysis. For animal experiments, the recombinant L. lactis (1×10^ 10 CFU/mL, 100 μL/dose) were administered via oral gavage daily for 7 consecutive days, followed by a booster immunization after a two-week interval to complete the oral vaccination protocol. The colonization capability of recombinant L. lactis in mouse intestines, its M cell-targeting specificity, and vaccine immunogenicity were systematically evaluated. Subsequently, protective efficacy assessments were conducted against both streptozotocin (STZ)-induced type 1 diabetes (T1D) in BALB/c mice and spontaneous non-obese diabetic (NOD) mouse models. Results: 1. The recombinant L. lactis vaccines CGIH, HGIC, GIH, and SGIH were successfully constructed, and an oral dose of 100 μL (1×10^ 9 CFU/mL) demonstrated effective induction of T1D tolerance. 2. L. lactis strains carrying the C and S sequences exhibited intestinal colonization levels tens of times higher than those lacking these sequences (nearly 40-fold increase in BALB/c mice and 60-fold increase in NOD mice). 3. Oral administration of the recombinant L. lactis-based targeted vaccines established an immune-tolerant niche for T1D, delaying disease progression. Notably, the HGIC vaccine, with the C sequence positioned after the GI motif, exhibited superior efficacy. 4. Following anti-CD25 monoclonal antibody-mediated Treg cell depletion, the targeted recombinant L. lactis vaccines stimulated Treg cell proliferation and differentiation, alleviating pathological damage caused by Treg cell exhaustion. Conclusion: Four recombinant L. lactis vaccines expressing immunogenic GAD65191-320 and INS17-110 fusion proteins were successfully constructed. CGIH and HGIC vaccines demonstrated intestinal M cell-targeting specificity and protective effects in both STZ-induced BALB/c and NOD T1D models. This study provides theoretical insights for the development of therapies and vaccines against T1D and other autoimmune diseases.