Expression Of Recombinant Human Proinsulin Using Oleosin Fusion Expression Systems In Brassica Napus

Diabetes is a group of metabolic diseases, which is caused by the absolute or relative secretes inadequacy of in vivo insulin, characterized by hyperglycemia and effected by environment and inherit. The complications of diabetes include cerebral apoplexy, ablepsia, amputation and so on, which are caused mostly by macrovassular complication and microangiopathy. The human health and the life quality are affected heavily by diabetes and the complications of it. Today, insulin is the most effective and safe first-line drugs to cure diabetes in clinical. Along with the number of clinical patients rising continuously, the high-speed growth market demand of insulin medicaments has increased year by year. The production and research of insulin display broad prospects for application and development.Since the first diabetic was cured successfully by insulin in 1922, the clinic application of insulin has a history of almost 90 years. Currently, insulin medications are mostly extracted from animal pancreas or produced by microbial fermentation for clinic, which have many disadvantages such as complexity of in vitro processing, high cost, low pureness purity and easy to pollution. The plant expression system of pharmaceutical proteins is a safe and cheap product system, which is attached importance to progressively. Plant oil body expression has improved the afterward process of plant bioreactor and reduced the cost of high purity pharmaceutical proteins, which make use of the high expression and easy-separation of the oleosin. Oil body expression become the new source of pharmaceutical proteins and will be perfected and widely used stage by stage.This study tried to produce recombinant human proinsulin in Brassica napus, which provide a new ways to therapies for clinical diabetes and has made the necessary preparation of techniques and theoretics for the other valuable pharmaceutical proteins produced in plant oil body expression. The results were summarized as follows:1. Gene synthesis and Vector construction: The oleosin promoter and oleosin gen(eBN-V) were cloned from huyou15(Brassica nape cv. Huyou 15). The hmins gene was designed and synthesized according to plant codon usage preference, which was connected to the oleosin gene downstream. In order to easily test and purify the heterologous protein, a His-tag(six histidine codons) was introduced in the front of oleosin gene(BN-V). Subsequently, the plant expression vector op::his-og-mins-2300-twin was successfully constructed.2. Preparation of engineering strain EHA105: The vector op::his-og-mins-2300-twin was introduced into Agrobacterium tumefaciens strain EHA105 by freezing-melting method, and we successfully obtained engineering strain EHA105, and positive clones were confirmed using PCR technique.3. Genetic transformation of Brassica: The resulting vector was introduced into Brassica via A.tumefaciens-mediated transformation. 160 putative transgenic tobacco plants were regenerated through kanamycin(40mg·L-1) selection. PCR screening showed that 8 transgenic Brassica plants were confirmed primary that the positive rate was 5%.4. Gene integration: Southern blot analysis showed that 6 independent transgenic Brassica plants were obtained. The target gene was integrated into the genome of transgenic Brassica plants, and ranging from 2 to 5 copies.5. Transcription expression: Semi-quantitative RT-PCR results indicated that the target gene hom was expressed at transcription level in mature seeds of 3 independent transgenic Brassica lines.6. Protein expression: The Western blot results showed that the target protein was expressed in 2 transgenic Brassica lines.7. Expression content: ELISA analysis showed that the percent of the goal protein in total soluble protein of Brassica mature seeds was 0.0179%, and 0.25% in oleosin protein of Brassica mature seeds.The plant oil body expression vector was constructed in this study and successfully expressed in Brassica mature seeds. With the seeds containing oleosin-proinsulin fusion collected, further purification and activity studies will be available.