1Expression And Activity Analysis Of Single Domain Antibody Fragments For The Fc Region Of Mouse IgG And Alkaline Phosphatase2Research On Transformation Of E.coli Genetic System

1Research on expression and application of VHHs and APThe single domain antibody is formed only by one domain-the variable region ofheavy chain of heavy-chain antibody, which is composed only of heavy chain.Compared to the conventional antibodies and single chain fragment variable (scFv),VHHs have several advantageous properties, such as the small size, good solubility,high stability and easy production as recombinant protein. These unique beneficialproperties make them useful in fundamental research. In this study, the gene of vhhsand ap have been cloned and expressed. Then, the external expression conditionswere optimized and the target proteins were purified. At last, the affinity of VHHs andenzyme activity of AP were determined. These results were laid a foundation forappliaction. The main contents are illustrated as following:1.1Cloning and expression of VHHs and APThe target proteins were expressed as soluble form in plasmid pET25b(+),E.coli Origami B through the analysis of the location of expression and the geneticbackground of bacteria.1.2Optimization of external expression conditions and purification of VHHs and APAfter the optimization of temperature of expression and liquid volume, theproteins were expressed at30℃with70%liquid volume. Then, the recombinantproteins were purified with HisTrapTMHP. The yield of recombinant protein T9, T10,T9-AP and T10-AP was50mg/L,10mg/L,26mg/L and6mg/L determined by BCAmethod.1.3The analysis of affinity of VHHs and specific activity of APBy ELISA method, the affinity constant of T9, T10, T9-AP and T10-AP was2.5×107L/M,6×107L/M,1.96×107L/M and2.96×107L/M. The results showed thatthe protein could meet the actual needs. According to the increase in OD405, thespecific activity of AP, T9-AP and T10-AP was224U/mg,34.85U/mg,36.12U/mg.The results showed that the activity of T9-AP and T10-AP was reduced because of the reduction of activity of AP.2Research on Transformation of E.coli genetic systemThe study of function of gene and construction of genetic engineering bacteriabasically rely on rapid and high-efficient gene knock-out method. The new methodwas based on site-specific recombination system and the principle of plasmidincompatibility. In order to improve the efficiency of gene recombination, weconstructed the new targeting vectors and screened the recombinant by twoantibiotics. Consequently, the efficiency of gene recombination attained100%. Thus,the establish of new method provides important insights into gene function researchand genetic engineering bacteria with new genetic characteristics...