High-Level Express Humanin Protein By Plant Oleosin Expression System

Alzheimers disease is a kind of common diseases in elders, which have the symptoms, such as, memory loss, cognitive dysfunction, became seriously with the increasing of old population. The patient’s quality of life have been affected seriously and it has become another serious diseases harm to human health after the disease of cancer, stroke and heart head blood-vessel.Humanin protein has a protective effect to the nerve cell damage caused by Alzheimers disease. Recent studies considered that Humanin has medicinal value in a wide range. It is a kind of important medicinal proteins with potential huge market demand. Plant bioreactor is an effective way in foreign protein production. It has many advantages, such as, low-cost, high-safety and so on.Various of enzymes and antibodies have been expressed so far. Foreign protein can be expressed on a high-level in plant seeds by the oleosin expression system. Oleosin has the double peculiarities of hydrophilic and lipotropy. It’s middle lipotropy region is very keep and it is very important to the localization in oil body, but the sequences of its C and N terminal are very difference, when foreign proteins insert to the ends, the localization and expression of the oleosin in oil body will not be affacted, which benefit to the foreign protein’s separation and purification. In this research, we used peanut oleosin expression system expressing Humanin protein, seek new plant bioreactor carrier, optimize the expression condition, for agriculture, genetic engineering and biomedical research is of great significance.In this study, we established peanut oleosin expression system to express Humanin protein primarily, and expressed in the tobacco seeds, lay the foundations for the Huamnin proteins production in peanuts. The main research contents and results are as follows:Synthesis of Humanin gene:We designed and synthesised two complementary single-stranded DNA primers (with corresponding enzyme loci and modifying sequence respectively), obtained right cloning of Humanin gene by cloning to the T vector and sequencing.The construction of plant expression vector:connect the humanin gene with peanut oleosin gene3’end, they were connected by coding sequence of erepsin, and drived by35s promoter or peanut oleosin promoter respectively. Plant expression vector pCAMBIA2300-oleopromoter-Aholeosin-Humanin and pCAMBIA2300-35S-Aholeosin-Humanin have been structured; Builded express unit3’of oleopromoter-Aholeosin-Humanin to the pCAMBIA1301vector which containing GUS, have obtained the plant expression vector pCAMBIA1301-oleopromoter-Aholeosin-Humanin-GUS; Builded express unit3’ of oleopromoter-Aholeosin-Humanin to the pCAMBIA1302vector which containing GFP, have obtained the plant expression vector pCAMBIA1302-oleopromoter-Aholeosin-Humanin-GFP.Transformation and Humanin expression in tobacco:Plants expression vector with purpose gene were infected tobacco by the method of agrobacterium mediated, regeneration plants were screened in the medium containing corresponding antibiotics. For the transformant of two expression vectors, pCAMBIA2300-oleopromoter-Aholeosin-Humanin and pCAMBIA2300-35S-Aholeosin-Humanin, DNAs were extracted from the leaves of tobacco plants, and positive transgenic tobacco lines were tested by PCR amplification. Using the RT-PCR method, the humanin expression from RNA transcriptional level was detected. As for the other two vectors, transgenic plants seeds of T1generation were dyed and made GFP investigation derictly in order to detect the expression of foreign proteins.The results are as follows:Four carriers have obtained the positive plants with25strains respectively. Foreign protein also can express highly in the RNA level by the result of RT-PCR. Using the method of GUS test, it was proved that foreign protein can express highly in the seeds. So far, we have obtained33strains homozygotes by screening the transgenetic offspring generations, illustrated peanut oleosin system can express the foreign protein effectively as a plant bioreactor system.