Vandenbroeck K, Fiten P, Beuken E, Martens E, Janssen A, Van Damme J, Opdenakker G, Billiau A
Rega Institute, University of Leuven, Belgium.
Eur J Biochem. 1993 Oct 1;217(1):45-52. doi: 10.1111/j.1432-1033.1993.tb18216.x.
A genomic clone (PIL3) covering the 8.8-kb prointerleukin-1 beta ('catabolin') gene of the domesticated swine (Sus scrofa domestica) was isolated from a genomic library and characterized by nucleotide sequencing. Typical features of the gene include a seven-exon structure, with the highest degree of nucleotide and amino acid conservation among human and porcine genes being found in the receptor-binding portion encoded by exons six and seven. Three 250-bp repetitive elements with a > 75% similarity to the pig repetitive element-1 family sequence are located in untranslated gene segments. Southern-hybridization experiments disclosed extensive genomic heterogeneity of the porcine interleukin-1 beta (IL-1 beta) gene region, suggesting a duplication of at least the 3' half of the gene in the porcine genome. Since similar hybridization patterns were observed for wild boar (Sus scrofa) genomic DNA, it was concluded that this gene rearrangement had preceded domestication of the wild swine. In addition, the cDNA for processed porcine IL-1 beta was constructed through polymerase-chain-reaction-mediated exon fusion by overlap extension starting from the genomic template. Recombinant IL-1 beta was expressed in Escherichia coli as a fusion protein containing an N-terminal hexahistidine tag followed by a factor-Xa-cleavage site. The protein was efficiently purified through adoption of a scheme that consisted of four alternating cycles of immobilized metal-ion-affinity chromatography and size-exclusion chromatography. 13.8 mg highly purified recombinant porcine IL-1 beta was obtained starting from a 900-ml thermo-induced E. coli culture (final endotoxin concentration < 0.22 ng/ml). The protein behaved homogeneously as a monomeric species, which was reactive in Western-blot experiments with an anti-(human-IL-1 beta) serum and which appeared to induce gelatinase B in MDBK cells in a dose-dependent fashion.
从基因组文库中分离出一个覆盖家猪（Sus scrofa domestica）8.8 kb前白细胞介素-1β（“分解素”）基因的基因组克隆（PIL3），并通过核苷酸测序进行表征。该基因的典型特征包括七外显子结构，在由外显子六和七编码的受体结合部分中，人与猪基因之间的核苷酸和氨基酸保守程度最高。三个与猪重复元件-1家族序列相似度> 75%的250 bp重复元件位于非翻译基因片段中。Southern杂交实验揭示了猪白细胞介素-1β（IL-1β）基因区域广泛的基因组异质性，表明该基因在猪基因组中至少3'端一半发生了重复。由于野猪（Sus scrofa）基因组DNA观察到类似的杂交模式，得出结论，这种基因重排在野猪驯化之前就已发生。此外，通过从基因组模板开始的聚合酶链反应介导的重叠延伸外显子融合构建了加工后的猪IL-1β的cDNA。重组IL-1β在大肠杆菌中作为融合蛋白表达，该融合蛋白包含N端六组氨酸标签，其后是因子Xa切割位点。通过采用由固定化金属离子亲和色谱和尺寸排阻色谱四个交替循环组成的方案，有效地纯化了该蛋白。从900 ml热诱导的大肠杆菌培养物中获得了13.8 mg高度纯化的重组猪IL-1β（最终内毒素浓度< 0.22 ng/ml）。该蛋白作为单体表现出均一性，在Western印迹实验中与抗（人IL-1β）血清反应，并似乎以剂量依赖方式在MDBK细胞中诱导明胶酶B。
