Fusion Expression And Antibacterial Activity Of Bovine Lactoferricin

Background and Objective:With the rapid economic development,people are increasingly concerned about their own lives and health because food safety is closely related to people’s livelihood.The food production,preservation and transportation are inseparable from the use of preservatives.Currently,chemical preservatives are widely used in the places of market,restaurant and so on.However,the long-term consumption of chemical preservatives is harmful to human life and led to a potential “triple” effect in the end.Meanwhile,the exploitation of new green and environmentally synthetic chemical preservatives is more and more difficult.As a result,people began to pay their attention to the natural preservatives that exist in nature.Bovine Lactoferricin(LfcinB)is a small peptide containing several tens of amino acid residues produced by degrading the N-terminal of lactoferrin(Lf)by acid pepsin.It has a broad-spectrum bactericidal capacity,which can inhibit the growth of fungi and bacterial.Moreover,lactoferricin have a series of biological activities,such as antivirus,anti-cancer,anti-oxidation,and regulation of immune function.It is noted that lactoferricin is not contain rare amino acids and its molecular weight is very small.They are also easily absorbed and degraded and have no side effects on the human body.Therefore,lactoferricin has broad application prospects in medical,food,feed,preservatives and many other fields and it has become a hot spot in current research and development.Method:We are designed a column-free method for the purification of heat-stable singlestranded DNA binding proteins and uses TaqSSB protein as a tag,which can fusion and expression purification of LfcinB.This method does not depend on the column and does not need to add other affinity.And target protein is mainly purified by heating,ammonium sulfate salting-out,and enzymolysis.1)Three expression vectors,pET22b-TaqSSB,pET22b-LfcinB-TaqSSB(N)and pET22b-TaqSSB-LfcinB(C).2)Design a column-free purification scheme for TaqSSB protein.And we conduct the experiment of column-free purification of TaqSSB protein based on the interaction between TaqSSB characteristics,ammonium sulfate salt concentration,heat-resistant temperature and heat-resistant duration.3)Design the fusion model of LfcinB protein and TaqSSB protein.The columnless purification protocol of TaqSSB protein was coducted to express and purify the fusion protein.4)Screening pepsin for fusion protein conditions and purifying LfcinB only by heating and centrifugation5)Identification of LfcinB by using High Performance Liquid Chromatography,Mass Spectrometry and Peptide Amino Acid Sequencing.6)Identify the antibacterial activity of LfcinB in Vitro.Results:1)An engineered strain that expresses TaqSSB protein efficiently was constructed and heated at 80°C for 20 minutes under 40% saturation salting conditions.Columnfree purification of TaqSSB protein resulted in the purity of TaqSSB protein exceeding 90%.2)Two fusion engineered bacteria were designed and constructed,and they were purified by heating at 80°C for 20 min under salting conditions of 40% saturation.These two fusion proteins can be purified to a purity of over 90%.3)The reliability of LfcinB obtained by this method was identified from the three aspects,including molecular weight level,structure sequence and antibacterial activity.The results showed that the effective components of bovine lactoferricin and other peptides in the purified hydrolysate were up to 68.96%,and had a certain antibacterial effect against Gram-positive and negative bacteria,and Gram-positive bacteria were more sensitive.The column-free purification method proposed in this topic has the advantages of simple operation,short time,and low cost for the LfcinB product.