美洲大蠊i型溶菌酶的原核表达及多克隆抗体制备

Prokaryotic Expression of i-type Lysozyme from Periplaneta americana and Preparation of Its Polyclonal Antibodies

旨在获得美洲大蠊i型溶菌酶Pa I的原核表达蛋白,并制备鼠抗Pa I多克隆抗体。PCR扩增Pa I成熟肽编码序列,构建原核表达载体p ET28a-Pa I,诱导表达、纯化Pa I-His融合蛋白,免疫Balb/c小鼠,间接ELISA法检测抗血清效价,Western blotting检测多克隆抗体的特异性。结果显示,Pa I成熟肽部分的编码序列长414 bp,由137个氨基酸组成。构建的原核表达载体p ET28a-Pa I在大肠杆菌Rosetta(DE3)中成功表达,SDS-PAGE检测显示纯化的Pa I-His融合蛋白的大小约为18 k D,间接ELISA和Western blotting分析表明免疫小鼠产生的抗血清具有较高的效价和较强的特异性。美洲大蠊溶菌酶Pa I成功实现原核表达,并获得了高效价特异性多克隆抗体。

The objective of this work is to express i-type lysozyme（Pa I）from Periplaneta americana in Escherichia coli and prepare its polyclonal antibodies of anti-Pa I in mice. The coding gene of mature peptide region was amplified by PCR and prokaryotic expression vector p ET28a-Pa I was constructed. Pa I-His fusion protein was expressed with IPTG induction and purified by Ni-NTA affinity chromatography. Then, Balb/c mice were immunized with the purified recombinant protein, and the titers of antiserum and the specificity of polyclonal antibodies were detected by indirect ELISA and Western blotting respectively. Results were as below. The length of nucleotide sequence encoding the mature peptide was 414 bp that encoded a putative protein with 137 amino acids. The constructed prokaryotic expression vector p ET28a-Pa I was successfully expressed in Escherichia coli. SDS-PAGE detection indicated that the Pa I-His fusion protein was about 18 k D. Indirect ELISA and Western blotting analysis showed that the antiserum from immunized mice had high titer and specificity. In conclusion, the prokaryotic expression of Pa I protein was successfully realized, and the anti-Pa I polyclonal antibody with high efficiency and specificity were prepared, which laid the foundation for the further researches on the biological function of Pa I protein.