Objective:To screen the new candidate molecules interacting with protein kinase Wee1B by yeast two hybrid system, and to analyze their interaction with Wee1B in the early stage of mouse fertilized eggs by bioinformatics.Methods:The plasmid pcDNA3.1/V5-His-TOPO-Wee1B wild type encoding mouse Wee1B gene was used as template to construct bait plasmid pGBKT7 Wee1B and the bait plasmid pGBKT7-Wee1B was transformed into yeast competent cells at SD/Trp (SDO),SD/Trp/X-α-Gal (SDO/X)and SD/Trp/X α Gal/AbA plates (SDO/X/A)plates to detect the toxicity and self-activation ability of yeast and its expression in yeast using Western blotting method.The yeast cells containing pGBKT7-Wee1B were fused with human ovary cDNA library, the yeast plasmid transformation of Escherichia coli positive clones were sequenced after identified by yeast transformation.BLAST analysis was carried out in GenBank,and its effect on the development of mouse fertilized eggs was deduced according to the gene annotation.Results:The double enzyme digestion analysis and sequencing analysis results showed that the pGBKT7-Wee1B bait plasmid was successfully constructed.The plasmid was transformed into the yeast,and there were no clones in the SDO/X/A plates.The pGBKT7-Wee1B and pGBKT7 empty vectors were transformed into the yeast,the bacteria were inoculated on the SDO plates,and the clones were uniformly grown on the two SDO plates.The positive clones were picked and expanded in culture,the protein was extracted and Western blotting showed that pGBKT7 Wee1B was expressed in the yeast.The bait plasmids were fused with human ovary cDNA library and the positive clones inserted into the fragment were identified by PCR. Nine proteins which interacted with Wee1B protein kinase were screened out by sequencing and blast analysis,and the proteins which could be closely related to the development of mouse oocytes and the development of fertilized eggs were analyzed by bioinformatics analysis.Conclusion:Using the yeast two hybrid system from human ovary cDNA library,nine interacting proteins with Wee1B protein kinase are screened and these screened proteins may regulate mouse oocyte maturation and early embryo development through interacting with Wee1B.

Phenylalanine hydroxylase (PAH) is a member of aromatic amino acid hydroxylase (AAAHs) family, and catalyze phenylalanine (Phe) into tyrosine (Tyr). Using immunological and RT-PCR methods to prove the existence of phenylalanine hydroxylase (PAH) gene in the brain of Neanthes japonica in protein and nucleic acid level. Using Western blotting to detect the pah immunogenicity of Neanthes japonica. Making paraffin sections and using immunohistochemical technique to identify the presence and distribution of the phenylalanine hydroxylase gene in the brain of Neanthes japonica. Clone pah gene from the brain of Neanthes japonica by RT-PCR, constructing plasmid and transferring into E. coli to amplification, picking a single homogeneous colony, double digesting then making sequence and comparing homology. Western blotting results showed that the expression of the protein is present in Neanthes japonica brain, immunohistochemistry technique results showed that phenylalanine hydroxylase mainly expressed in abdominal of forebrain, dorsal and sides of midbrain. RT-PCR technique results showed that the phenylalanine hydroxylase exist in the brain of Neanthes japonica and has a high homology with others animals. PAH is present in the lower organisms Neanthes japonica, in protein and nucleic acid level. Which provide the foundation for further study the evolution of aromatic amino acid hydroxylase genes in invertebrate.
Animals ; Blotting, Western ; Brain ; enzymology ; Escherichia coli ; metabolism ; Phenylalanine Hydroxylase ; genetics ; metabolism ; Polychaeta ; enzymology ; genetics