Construction Of The Vector With Serious Parotin Gene And Parotin Expression

Water from the margin of the tongue equals to“huo”, in the Chinese writtenlanguage, meaning the tongue side of water (saliva) to maintain the body’s life activity.Saliva is the mixture secreted by the oral salivary gland, a kind of foam liquid whichis colorless, tasteless nearly neutral. Its composition is similar with plasma. Most partof saliva is water, in addition is a variety of salt ions, enzymes, immunoglobulins,nerve growth factor, also includes a endocrine protein hormone--Parotin.Parotin is mainly composed of parotid gland, submandibular gland secretion.Parotid and submandibular gland secretion of saliva in the mouth before flowing intothe discharge pipe, the gland "fringe" flow is slow, parotin, a kind of endocrinehormone in saliva get absorbed in here. People over20years old, salivary glandsbegin to fatty atrophy, secretion decreased. With the increase of age, the symptom isaggravating gradually, until more than60years old, parotid gland tissue will bereduced to the original60%. People entering the mature stage, comparing tosubmandibular gland, sublingual gland, parotid acinar atrophy obviously earlier,replaced by fat. The changing structure of glandular stripes, reduced the parotinabsorption.Parotin deficiency can result in mesenchymal tissue dependent disorder caused bythe false phenomenon of aging, this is secondary aging and not really aging, ischanged from the early old age increasing to elderly sick. False aging may causecartilage softening, osteoporosis, alveolar pyorrhea etc. Cartilage softening, is one ofthe reasons why the deforming arthritis or vertebral disease. Osteoporosis is that boneis thin, crisp and bone trabecula sloppy. If in the development stage, lack of parotin,there would be a tendency of short limb disease. Alveolar pyorrhea is embodied in theperiodontal tissue change, gum edge with severe inflammatory infiltration. Parotin oninduced mesenchymal tissue nutritional disorders (false aging) due to lack of parotinhas curative effect. Parotin is a protein hormone, whose level extremely in saliva is low, and theseparation is very difficult. Chemical synthesis of parotin, whose amino acid may beracemic, and the cost is high, not suitable for mass production.Genetic engineering is an important way of modern biological science to obtainthe target protein. Because of low molecular weight, construction of single parotingene recombinant vector for expression using conventional genetic engineeringmethod, the expression level is very low and the molecular is unstable. In thisexperiment I adopted tandem gene technology to connect parotin gene directed andincrease its copy, so the expression quantity and molecular stability are greatlyimproved.First, we use SOE-PCR to obtain full-length of parotin gene, we respectivelyadded the recognition sites of isocaudamer enzyme XhoI, SalI and the cleavage sitesof cyanogen bromide, hydroxylamine on primer5’end. We can operate the gene andthe cloning vector with isocaudamer enzyme to constructs the recombinant vectorwith9copy of parotin gene gradually.Then we chose pET32a as the expression vector to express the parotin geneconcatemer and labels on the vector together. The molecular weight of the expressedprotein is about35KD. Through the SDS-PAGE we detected the protein existing, andthen by Western Blot and His-Tag antibody, we identified the parotin. Optimizing theexpression conditions, we expressed with0.5mM IPTG for after3hours at37℃, andextracted the total bacterial proteins. We obtained the series of patotin protein afterpurification by Ni Sepharose, and lay the foundation for the following research.