The Construction Of Bactericidal Permeablility-Increasing Protein And Defensins Fusion Plasmid Of Cattle And Its Expression In E. Coli

1. Objectives and SignificancesWhen the organism is attacked by external pathogens, Living organisms themselves will start their active defense system to resist invasion. Neutrophil is an important component in this active defense system. Neutrophil possesses a variety of anti-microbial activity of proteins and peptides, including bactericidal/permeability-increasing protein and defensins. In the research of mastitis disease, it was found that P-defensin genes could be induced to express in galactophore of mastitis cows. This Phenomenon indicates that they possibly participate in part defense of galactophore. Meanwhile, defensins had anti-gram-positive bacteria(G+) and anti-gram-negative bacteria(G-) biological activity. in other words, defensins had inhibited function on Staphylococcus Aureus and Escherichia Coli, which are the major mastitis pathogens in cows. Therefore, bovine defensins could be candidate genes of mastitis for further study. The research is of great significance on the prevention and against diseases of mastitis. In addition, in the process of dariy cattle production, endotoxin-induced disease complications are still not very effectively treated. However, BPI possibly have the ability to treat the endotoxin-induced disease. At the same time, we found that BPI has a specific anti-gram-negative capacity, just to make up deficiencies for the defensins in the anti-gram-negative capacity. BPI and defensins are not easily produced bacteria drug resistance. Therefore, we hope to construct a BPI and defensins fusion protein by using gene engineering technology, in order to combine their biological activity and make a new antibacterial agent which has wider antibacterial spectrum and stronger antibacterial activity, so as to can be used in the treatment of more diseases. In addition, the fusion protein also could exactly solve the problem that small molecular peptides or proteins are easily degradated due to their short halflife. Moreover, it also may increased the gene production. Therefore, construction of the BPI and defensin vector and its expression is a very meaningful and valuable work.2. Methods and Results(1) This experiment using the blood of Hostein cow which troubled with mastitis. Total RNA was extracted from leucocytes of blood. According to the GenBank bovine BPI gene sequence (Login:BC102310.1, GI:74354930) primers was designed by Primer Premier 5.0, in which EcoRâ… and Salâ… restricted enzyme sites were added at the end of 5'. By using RT-PCR technology and Nested PCR methods the N-terminal cDNA sequence of BPI gene was amplified. cDNA fragment was approximately 714bp. Then, it was cloned into pEASY-T1 vector, the plasmids that had been sequenced proved to be right named as pEASY-T1-BPI. BLAST result showed that the nucleic acid sequence homology was 99%, however the amino acids sequence homology was 100%. According to bovine BNBD4 GenBank sequence (Login:NM₁74775.1 GI: 28849940), primers was designed by Primer Premier 5.0, in which Hind III and Xho I restricted enzyme sites were added at the end of 5'. By using RT-PCR technology the cDNA sequence of BNBD4 gene was amplified. cDNA fragment is approximately 192bp. Then, it was cloned into pBluescript II SK (+) vector, the plasmids that had been sequenced proved to be right named as pBluescript-BNBD4. BLAST result also showed that the nucleic acid sequence homology was 100%.(2) First, The empty pET-28 (a+) plasmid was linearized by restriction enzyme EcoR I and Sal I.Then, linked with the BPI gene. The plasmids that had been sequenced proved to be right named as pET-BPI. The pET-BPI plasmid was linearized by restriction enzyme Hindâ…¢and Xhoâ… .Then, linked with the BNBD4 gene. The plasmids that have been sequenced proved to be right named pET-BPI-BNBD4.(3) Engineering bacterias that contain all recombinant plasmid pET-BPI-BNBD4 were induced gene expression by IPTG. SDS-PAGE results showed:37℃, IPTG 1.0mmol/L, the highest expression level when induced by 4 hours. The fusion protein was displayed in the both supernatant and in the inclusion bodies SDS-PAGE, the molecular weight of fusion protein is approximately 37.3kDa, according with size predicted by bioinformatics software ProtParam. Western blot result also showed that the location of the corresponding protein band appeared clearly.(4) Fusion protein in vitro antibacterial test:Agar-diffusion method and the Enzyme-labelled meta test results both manifested that this fusion protein had certain degree inhibited function on Staphylococcus Aureus and Escherichia Coli.3. ConclusionIn summary, the above data demonstrated that we successfully cloned BPI gene N-terminal and BNBD4 gene, constructed pET-BPI-BNBD4 fusion expression vector, and it was successfully expressed the fusion protein BPI-BNBD4 in Escherichia Coli. In vitro tests showed that this fusion protein had both G+(Staphylococcus Aureus) and G-(Escherichia Coli) biological activity. This research laid a foundation for the complete biological activity tests of the fusion protein in further research.