Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease characterized by the destruction of insulin-secreting β cells upon autoreactive T cell attack. Oral administration of autoantigens is an attractive approach to treating T1DM, but an effective carrier should be used in order to protect antigens. Lactococcus lactis, a safe engineering strain, was used for this task in the present study. Two recombinant L. lactis expressing protein HSP65-6IA2P2 were used and be investigated the effects and mechanisms against T1DM in NOD mice. Our findings demonstrate that recombinant L. lactis strains can successfully both deliver antigens to intestinal mucosa and maintain the epitopes for a long time in NOD mice. Oral administration of recombinant L. lactis could prevent hyperglycemia, improve glucose tolerance, and reduce insulitis by inhibiting antigen-specific proliferation of T cells, augmenting regulatory immune reactions, and balancing ratios of Th17/Tregs and Th1/Th2. These results prove that orally administrated L. lactis expressing HSP65-6IA2P2 is an effective approach for the prevention of T1DM in NOD mice.

Lactic acid bacteria are normal in human and animal intestinal which are generally recognized as safe microorganism.Lactococcus lactis is the mode bacteria of lactic acid bacteria and its genome has been sequenced,and using the molecular biology tools,it's easy for the modification of recipient bacteria which are suitable for all kinds of expression vector.Currently it has been developed that a series of plasmid cloning vector,integrated cloning vector system and protein localization system,meanwhile a variety of pathogenic microbial antigens have successfully expressed in Lactococcus lactis.The application situation of Lactococcus lactis in vaccines presenting carrier in recent years have been described,and elaborating research progress in the anti-infective,anti-cancer,anti-autoimmune aspects and difficulties encountered in vaccine development and the prospect are well explained.

To study the hypoglycemic effect and mechanisms of vaccine HSP65-6P277 protein on type 1 diabetic mice induced by streptozotocin( STZ),Lactococcus lactis live vector was used to express the vaccine protein. The mouse model of type 1 diabetes mellitus were established by intraperitoneal injections of multiple low dose STZ( MLD-STZ) and divided into three groups. The inducible expression strain L. lactis NZ9000 pCYT ∶ HSP65-6P277,constitutive expression strain L. lactis NZ9000 pHJ ∶ HSP65-6P277 and control strains L. lactis NZ9000 pCYT ∶ Nuc were respectively immunized to diabetic mice orally once daily( 2 × 109CFU,200 μL) in the first week and once every week in the following weeks lasting for 16 weeks. Fasting blood glucose levels were measured once a month on blood samples taken from orbital venous plexus. Four months later,the mice were sacrificed. Spleen cell proliferation experiment was performed. IL-10 and IFN-γ cytokine levels of spleen cells and serum IgG levels were assayed using ELISA kits. The results showed that pCYT ∶ HSP65-6P277 group and pHJ ∶ HSP65-6P277 group have low blood glucose( P 0. 05),and maintain normal body weight,compared with control group( pCYT ∶ Nuc). On the other hand,cytokine IFN-γ levels were significantly lower( P 0. 05) in spleen cells supernatant and spleen cell proliferation was reduced against HSP65 and P277( P 0. 05),but serum IgG levels were not significantly different( P 0. 05).

IA-2-P2是由来自P277多肽的T表位与胰岛素瘤相关蛋白2(IA-2)的B表位组合而成的新疫苗肽,为验证其对1型糖尿病的治疗效果,采用多次小剂量注射链脲佐菌素(STZ)诱导C57BL/6雄性小鼠建立1型糖尿病模型,成模2w后,sc 100 μg多肽进行免疫治疗,间隔3 w免疫,共6次,每3 w收集被免疫动物的血清进行生化分析,测定血糖浓度和IgG抗体滴度并进行体重监测.结果显示IA-2-P2多肽能在一定程度上降低血糖,与对照组相比,IA-2-P2组终末平均血糖下降38％;并能维持小鼠的体重的稳定;多肽治疗能延长小鼠生存时间,在32 w观测期间,IA-2-P2组小鼠生存率达70％,较对照组40％的生存率有显著差异(P＜0.05);并且具有免疫调节作用,ELISA结果显示多肽可以有效引起免疫应答产生,与对照组相比,抗体水平有显著差异(P＜0.05).

The HIS tag purification system is used to improve the production of recombinant protein and to simplify production technique.HSP65-6P277 was a kind of recombinant protein which prevented the type 1 diabetes.Here,a study with a purpose of high level expression and convenient procedure of this vaccine in Escherichia coli was presented.The 6 × His-tag was artificially added at HSP65-6P277 N-terminal using PCR(polymerase chain reaction) amplification technique.Then this gene cassette was cloned into pET28a and the HIS affinity tagged HSP65-6P277 fusion protein expression plasmid pET28a-HIS-HSP65-6P277 was successfully constructed.By lactose induction,the fusion protein HIS-HSP65-6P277 was expressed as soluble form in the E.coli BL21(DE3).The recombinant HIS-HSP65-6P277 was purified only by HIS Bind Resin affinity chromatography.The electrophoretic behavior in SDS-PAGE and the result shown in the Weston blotting illustrated that the purified HIS-HSP65-6P277 was a HIS tag fusion protein with molecular weight of 70 k.As a result,the time period of the whole production technique was reduced from 15 ～ 20 days to 2 ～ 3 days.And the production was increased from 12 mg to 56 mg per liter culture.In conclusion,the HIS tag purification system provides a novel approach to simplifying the production technique and amplifying production of a vaccine against type 1 diabetes.