Bioactivity Testing Of Recombinant IL-1β And Purification Of Serum IgM In Acanthopagrus Latus

Interleukine-1β(IL-1β) gene of the Acanthopagrus latus has been cloned in our lab.The sequences encoding predicted mature peptide were subcloned into expression vectorpQE30 and subsequently were transformed into E. coli M15. The further research on thebioactivity of IL-1βin this thesis was on this foundation.After IPTG inducement, the recombinant protein has been obtained in inclusion.The recombinant protein was purified with Ni²⁺-Chelating Sepharose FF chromatographyand the renaturation of recombinant protein was through gradient dialysis. The results ofsequencing and Western Blot can prove that the recombinant protein is the recombinantInterleukin 1β(rIL-1β). The purity of purified rIL-1βreached 95ï¼…by the analysis ofBand Scan5.0. The concentration of purified rIL-1βwas determined by Bradford'smethod and the result was 0.18mg/ml. Tachyplens Amebocyte Lysate assay revealed that theendotoxin content in the purified rIL-1βwas less than 0.964EU/ml.The head kidney leukcocytes of the Acanthopagrus latus were isolated using adiscontinuous density Percoll gradient centrifugation. It showed that the density of1.080g/ml was suitable for the isolation of head kidney leukcocytes and the percentage ofleukcocyte could exceed 95ï¼…. The leukcocytes were incubated with rIL-1βat 23℃for 4hours prior to total RNAextraction. The RT-PCR revealed that rIL-1βcould up-regulatethe transcription of IL-1βat 20ng/ml and the effect could be equal to 5μg/ml LPS.The serum IgM of Acanthopagrus latus was purified by Sepharose-6B gel filtrationchromatography, Phenyl Sepharose FF hydrophobic interaction chromatography andrProtein A Sepharose affinity chromatography. These methods were compared in severalrespects. The results of SDS-PAGE were analyzed by Band Scan5.0. The analysisindicated that the purity of the serum IgM attained by Sepharose-6B gel filtrationchromatography or Phenyl Sepharose FF hydrophobic interaction chromatography wasonly about 56ï¼…. Taking the combination of Sepharose-6B gel filtration chromatographyand Phenyl Sepharose FF hydrophobic interaction chromatography the purity couldapproach 69ï¼…. However the IgM could reach a purity of approximate 89ï¼…just usingrProtein A Sepharose affinity chromatography. Although the rProtein A Sepharoseaffinity chromatography revealed the excellent performance comparing with othermethods in this research, the expensive price limited its widespread application. Sepharose-6B gel filtration chromatography and Phenyl Sepharose FF hydrophobicinteraction chromatography could supply a good balance between price and performance.The analysis also showed that molecular weights of the heavy-chain and light-chain ofIgM were respectively 73.6 KDa and 26.SKDa. The IgM purified by rProtein ASepharose affinity chromatography was used to prepare rabbit sera anti-IgM The indirectELISA revealed that the titers of anti-sera reached 1:25600. These laid the foundation offurther researches on the adjuvant effect of rIL-1β.