Identification And Screening Of Genetic Engineering Antibodies Of Extracellular Alkaline Phosphatase PhoX In Microcystis

The problem of cyanobacteria blooms with Microcystis as the dominant species has attracted much attention.Phosphorus is an essential nutrient for the growth of Microcystis,so it is necessary to understand the process of phosphorus metabolism.Organophosphorus in water is an important phosphorus source of cyanobacteria,and extracellular alkaline phosphatase(PhoX)plays a key role in this metabolic process.The PhoX of Microcystis has not been confirmed by research so far.Accurate quantification of Microcystis PhoX is also an important link in clarifying the phosphorus metabolism process in the water of a bloom outbreak.In this study,based on the genome of Microcysits aeruginosa M.aeruginosa PCC7806(M.aeruginosa PCC7806),a possible extracellular alkaline phosphatase phoX gene(Accession no.ARI79942.1)was found,and the gene was expressed in E.coli,and then a series of enzymatic properties of the expression of the enzyme were analyzed,the enzyme was confirmed;The expression response of alkaline phosphatase phoX and phoA in M.aeruginosa PCC7806 under different phosphorus conditions was investigated.Using the extracellular alkaline phosphatase protein of M.aeruginosa PCC7806 as an antigen,we screened specific genetic engineering antibodies from a commercial Camel-derived nanoantibody library based on phage display technology,which is an accurate and effective tool for quantitative development of PhoX.Specific research results are as follows:(1)By constructing expression vector,expressing and purifying Microcystis phoX gene,a series of properties of the protein were analyzed.Western-blot and SDS-PAGE results showed that the molecular weight of the target protein was about88k Da,which was consistent with the expected expression size.Bioinformatics analysis showed that the protein contained 735 amino acid residues.The?-helix of the secondary structure is mainly distributed at the nitrogen end.There are a large number of glycosylation and phosphorylation sites in the prediction of protein post-translational modification.The protein is hydrophilic and has no transmembrane domain,but has nitrogen terminal Tat-way signal peptide.All these are consistent with the characteristics of extracellular alkaline phosphatase.The phoX expressed exhibited phosphodiesterase and phosphomonesterase activities,confirming that the protein was alkaline phosphatase.The optimum temperature and p H value of the enzyme were 37?and 10.Mg²⁺as a coenzyme had the highest activity,followed by CO²⁺,Ca²⁺,Zn²⁺and Mn²⁺.Ni²⁺inhibited the enzyme.(2)In the training to the logarithmic phase M.aeruginosa PCC7806 BG11medium were transferred to no phosphorus(BG11(-P)),inorganic phosphorus(BG11)and organic phosphorus(BG11(-P)+AMP)medium culture,respectively in the algal cells from 0,2,4,6 h RT-q PCR experiments,analysis of extracellular alkaline phosphatase protein-coding genes phoX and intracellular alkaline phosphatase gene phoA expression and alkaline phosphatase activity as a whole.The results showed that phosphorus had a significant response to phoX expression in different environments.PhoX expression reached the highest level at 2 h and then decreased to a low level at 4 h when cultured in phosphate-deficient medium(BG11(-P)).(3)Gene analysis showed that PhoX was highly conserved in Microcystis,which was different from other cyanobacteria.The PhoX protein of M.aeruginosa PCC7806 was used as the antigen,and the genetic engineering antibodies were screened from camel nanoantibody library based on phage display technology.After three rounds of screening,four VHH antibodies were finally obtained.VHH-3H,an 18k Da antibody protein,was obtained by purification of 3H with the P/N value of 10.35by affinity column expressing His-Tag.The FITC-VHH-3H antibody was labeled by FITC fluorescence,and the specific binding ability of FITC-VHH-3H antibody to Microcystis extracellular alkaline phosphatase was verified by immunofluorescence reaction.The specificity of VHH-3H antibody was verified by ELISA.The antibody can be used for the analysis of Microcystis alkaline phosphatase in water and the identification of Microcystis cells in environmental water samples.