Abstract: To study the biological functions of vitellogenin N domain（VitN） in Cherax quadricarinatus,and obtain VitN fusion protein with immune specificity, VitN mRNA sequence（coding 42-585 aa） was obtained according to the mRNA sequence of vitellogenin of Cherax quadricarinatus（GenBank accession number AF306784.1）. Bioinformatics analysis of VitN was carried out by online analysis tools. VitN gene sequence was optimized and synthesized to construct recombinant expression plasmid pET-28 a-VitN. The fusion protein was transformed into Escherichia coli BL21（λDE3） cells and induced by IPTG. The expression, purification, inclusion body renaturation, Western-blot identification and concentration determination of VitN fusion protein were performed. The results showed that there were 544 amino acids encoding VitN, all of which were common amino acids, among which alanine, valine and serine were the most, accounting for 7.72%, 7.72% and 7.54%, respectively. Tryptophan content was the least, accounting for only 0.92%. The relative molecular weight of VitN was 64 681; the theoretical isoelectric point was 8.69, a total of 8 540 atoms; the molecular formula was C₂₆₇₁H₄₂₆₀N₇₅₈O₈₁₈S₃₃, the instability coefficient was 31.49; the fat coefficient was 74.56, and the average hydrophilic value was-0.45. The number of hydrophilic amino acids was significantly higher than that of hydrophobic amino acids. The amino acids encoding VitN were all located outside the cell membrane without transmembrane region. VitN subcellular localization was extracellular or cytoplasmic examination. It did not contain conventional signal peptides, nor did it contain type Ⅰ signal peptidase. There were 67 phosphorylation sites in VitN, including 31 serine phosphorylation sites, 23 threonine phosphorylation sites, and 13 tyrosine phosphorylation sites. The secondary structure of VitN consisted of α-helixes, β folds, extended chains, and random curls. VitN contained lipid-binding lv-1 n and lv-1 c peptide chains. There were two obvious regions in the upper and lower parts of the tertiary structure. The upper part was enriched with sheet folding structure, and the lower part was enriched with α helix structure. There were two disulfide bonds at amino acids 129-153 and 396-402 respectively. The optimized VitN gene fragment size was about 1 650 bp. The fusion protein with a size of 64.7 ku was obtained by ligating, transforming and inducing. The main expression form of the fusion protein was inclusion body in E. coli BL21（λDE3） competent cells. When the OD₆₀₀ value reached 0.6, inducer IPTG was added at 250 r/min at 37 ℃ for 4 h and the final concentration was 0.5 mmol/L. When the inclusion body was purified by Ni-NTA column, the elution effect of 50 mmol/L Imidazole eluent was the best. The protein was further identified as the target protein by Western-blot. The final concentration of purified VitN fusion protein was 0.45 mg/mL. It indicated that bioinformatics analysis VitN in Cherax quadricarinatus was carried out; the recombinant expression plasmid pET-28 a-VitN was successfully constructed; the expression system and purification scheme were optimized, and the VitN fusion protein with immune specificity was obtained.