Abstract: 【Objective】 The objective of this study is to prepare the polyclonal antibody of Spodoptera exigua vitellogenin (Vg), investigate the expression pattern of Vg protein in female hemolymph of S. exigua at different developmental stages, and to provide a basis for studying the function and mechanism of synthesis, transportation and utilization.【Method】The fragment of Vg was amplified from the cDNA of 24-h-old female adults of S. exigua by PCR which included the vitellogenin-N region. The fragment of Vg was then inserted into the pMD-19T for sequencing. The nucleic acid sequence and the amino acids encoded by this gene fragment were analyzed by DNAMAN software. The sequenced Vg fragment was inserted into the expression vector pCzn1 by Nde I and Xba I digestion. The recombinant vector pCzn1-Vg was then inserted into Escherichia coli ArcticExpress. The E. coli ArcticExpress expressing the Vg recombinant protein was collected and crushed by ultrasonic. The supernatant and the precipitate were collected, respectively, and SDS-PAGE was used to analyze the expression of the recombinant protein. The recombinant Vg protein was expressed at different temperatures and concentrations of IPTG and the optimized expression condition was achieved. The recombinant protein was purified by Ni-NTA agarose. The purified recombinant protein was used to produce polyclonal antibody via immunizing rabbit. The titer of rabbit anti-Vg antiserum was evaluated by indirect ELISA. The content of Vg in female hemolymph of S. exigua at different developmental stages was detected by Western blot. 【Result】 The length of the fragment of Vg is 2 091 bp, encoding 697 amino acids. The predicted molecular weight of Vg recombinant protein is 80.88 kD. The molecular weight of Vg recombinant protein expressed in E. coli is 80 kD, which is consistent with the predicted molecular weight. It was mainly expressed in inclusion body rather than the supernatant. The results of Vg recombinant protein content expressed at different temperatures and concentrations of IPTG showed that it was highly expressed at inducing temperature of 25℃ with 0.6 mmol·L^-1 IPTG. It had no obvious effect on boosting the Vg recombinant protein level and other protein content increased by raising temperature and the concentration of IPTG. After immunizing New Zealand white rabbits with four times, the ELISA assay showed that the rabbit anti-Vg antiserum had a good sensitivity with the titer 1﹕512 000. The Vg content in female hemolymph of S. exigua at different developmental stages was detected by Western blot. A single Vg band of approximately 180 kD was detected. Vg was first expressed at late stage of female pupa and showed a low expression level. After female adult eclosion, Vg expression was in a dynamic balance which peaked in 48-h-old female adults, then decreased. 【Conclusion】 The Vg recombinant protein was successfully purified and the optimized expression condition (temperature of 25℃ with 0.6 mmol·L^-1 IPTG) is clearly defined. The polyclonal antibody of Vg protein with high titer was obtained and the expression pattern of Vg in S. exigua is explicit.

Key words: Spodoptera exigua, vitellogenin gene, prokaryotic expression, polyclonal antibody, expression pattern