Cloning, Prokaryotic Expression, And Preparation Of Polyclonal Antibody Of Serpin4 From Bombyx Mori

This investigation employed silkworm (Bombyx mori), strain Dazao as material and have cloned the serine protease inhibitor gene (serpin4) of silkworm utilizing cDNA derived from fat body of silkworm as template. Lipopolysaccharide (LPS) was utilized to stimulate the silkworm on the 3rd day of the 5th instar also their haemolymph and fat body were acquired on a certain time interval and the cDNA from the haemolymph and fat body was employed as template for quantitative analysis. E.coli prokaryotic expression system was used for expression study of serpin4 gene in vitro also the fusion protein of Serpin4 was purified and the purified fusion protein was used as antigen to immunize KM mouse for the sake of producing polyclonal antibody against Serpin4. The main research consequence are as follows:1. The primer was designed according to the references. Afterwards mRNA was extracted from fat body of Day-3 fifth instar "Daizo" larvae and the serpin4 gene without signal peptide sequence of silkworm (The GenBank accession:GU270470) was amplified by RT-PCR. The sequence analysis displayed that the coding region of the serpin4 gene without signal peptide sequence with a length of 1183 bp codes 393 amino acid and the molecular weight of the mature protein was about 44.5 kD.2. According to the acquired sequence of silkworm serpin4 gene, a pair of primers was designed for quantitative PCR reaction. The silkworm larvae on the 3rd day of the 5th instar were stimulated by LPS also the fat body and the haemolymph cDNA derived from the stimulated silkworm in distinct times was used for RT-PCR. The length of the PCR product was predicted as 268 bp. The silkwormActin3 gene was employed as internal reference (Sense (F):5'CTGCGTCTGGACTTGGC 3'Antisense (R):3' CGAGGGAGCTGCTGGAT 5') for quantitative actin3 results normalization and finally the relative expression level of this gene in different time periods was obtained by using the treatment group relative expression of the same time period divided by the relative expression of the control group.The results show that,serpin4 gene in the silkworm hemolymph after 6 h-12h after the induction first increased and then decreased. Serpin4 induced gene in silkworm fat body expression did not change significantly. It is reasoned that genes with silkworm Serpin4 immune function, but the gene may be involved in part of the immune response.3. The serpin4 gene without signal peptide was constructed to the prokaryotic expression vector pET-28a (+) and then transformed into E. coli BL21. And 1mmol/L IPTG was used to induce the target protein to express. SDS-PAGE analysis indicated that the serpin4 gene was expressed in the form of fusion protein.There was a specific band with relative molecular weight of 45 kDa on PAGE profiles for the BL21 with Serpin4 induced by IPTG,comparied to the contract BL21,BL21 with empty pET-28a (+) or BL21 with Serpin4 not induced by IPTG, which indicated that serpin4 gene was expressed in E. coli. Western blot analysis showed a obvious band on the expected position (45 kDa),in accordance with the molecular weight of the fusion protein, the specific band not detected for the other three group, which further confirmed that the Serpin4 gene without signal peptide was successfully expressed.4. After being induced by IPTG, we acquire prokaryotic expression recombinant fusion protein which was subsequently purified by Ni column. The molecular weight of the purified protein identified by SDS-PAGE and polyclonal antibody against His-tag was consistent with prediction, that is, acquiring interest protein with high purity, which was employed as antigen to immunize KM mouse by four separate immunizations and ultimately achieving polyclonal antibody serum against Serpin4.The acquired antibody was validated by Elisa and Western blot also its titer was 1:20000.The successful cloning, quantitative analysis of differential expression afte LPS infection, prokaryotic expressing research of serpin4 gene of Bombyx mori and the preparation of polyclonal antibody has further analyticed the relationship between Serpin and immunity of Bombyx mori.It also provides reference for studing immune defense of Bombyx mori and other Lepidoptera insects.