| Lipopolysaccharide(LPS) or endotoxin, which was one of the bacterial cell wall component, was the main morbigenous factor of gram-negative bacteria. Endotoxemia and correlative disease were the important death cause of human and animal. Endotoxin binding protein and Endotoxin binding protein receptors, especially lipopolysaccharide binding protein(LBP) and bactericidal/permeability increasing protein(BPI), played an important role in recognizing organism and regulating endotoxin function. BPI had an extensive bactericidal action for gram-negative bacteria and was considered as Zyvox in vivo. LBP had a very important relation with inflammatory reaction in organism. Which could combine with LPS, and then evoked inflammatory reaction via intracellular aignal transduction. In this reseach, We choiced BPI and LBP to study, for which could kill many kinds of gram-negative bacteria and neutralize bacterial endotoxin. We studied genetic variation of porcine BPI gene and LBP gene, cloned their functional fragment and preliminary study their functions. The result of this thesis were as follows:1. There were 12 SNPs and a 263bp insertion/deletion fragment in intron 10 of porcine BPI gene and 9 SNPs in intron 9 of porcine LBP gene. The length polymorphism of intron 10 in porcine BPI gene was detected by PCR. A length polymorphic segment in intron 10 is common in all detected groups. Alleles detected in the sequence were allele A and a with frequencies of 0.582/0.418 and 0.799/0.201 in the two populations, respectively. This polymorphic segment of BPI gene was at Hardy-Weinberg equilibrium (P> 0.05) in the two populations, respectively. Association between the founded length polymorphic segment in intron 10 and birth weight, body weight at 45 days, body weight at 60 days, body weight at 4 months, body weight at 6 months, body height at 6 months, body length at 6 months and circumference at 6 months was tested by least square analysis. No significant different between length polymorphic segment and birth weight, body weight at 45 days, body weight at 60 days, body weight at 4 months and body height at 6 months were founded in this study, but significant associations were found between the founded length polymorphic segment in intron 10 and body weight at 45 days, body weight at 6 months and circumference at 6 months (P<0.05) in different genotype body. The polymorphism of intron 9 in porcine LBP gene was detected by PCR-RFLP. A Msp I RFLP was detected in intron 9 and the polymorphic locus is common in all detected groups. Association between the founded Msp I RFLP in intron 9 and birth weight, body weight at 45 days, body weight at 60 days, body weight at 4 months, body weight at 6 months, body height at 6 months, body length at 6 months and circumference at 6 months was tested by least square analysis. No significant different between Msp I RFLP and birth weight, body weight at 45 days, body weight at 60 days, body weight at 4 months, body weight at 6 months, body height at 6 months, body length at 6 months and circumference at 6 months. Therefore, it could be seen that porcine BPI genotypes had significant influence on growth traits, this length polymorphic segment might be considered as a potential genetic marker for selection.2. In this study porcine BPI gene and LBP gene was cloned from porcine blood by reverse transcription-polymerase chain reaction(RT-PCR). Sequence analysis showed that the porcine BPI cDNA cloned was 1874 bp in length (GenBank accession No:FJ810853) and the open reading frame encoded 483 amino acids residues with 13.25% leucines, including a signal peptide of 27 amino acids. The comparison of amino acids sequence of porcine BPI with human, cattle, rabbit, dog, rat. mouse, carp, Xenopus laevis, atlantic salmon and large yellow croaker showed that the homology similarity were 64%,74%,59%,67%,53%,51%,35%,44%,28% and 27%, respectively. Between amino terminal and carboxyl terminal of the porcine BPI could be hydrolyzed by trypsase, which had distinct functional domains and transactivation domains. It shew the common structural features of human’s BPI. And the porcine LBP cDNA cloned was 1648bp in length and the open reading frame encoded 481 amino acids residues with 14.94% leucines, including a signal peptide of 25 amino acids. The comparison of amino acids sequence of porcine LBP with human, cattle, rabbit, deer, rat. mice and monkey, showed that the homology similarity were 74%,77%.66%,76%,63%.,64% and 73%, respectively. These results indicated that porcine BPI and LPB might play a crucial role in immune response and recognition of pathogen-associated molecular patterns and signal transduction.3. Specific primers, which contain specified restriction enzyme cutting site, was designed and its DNA sequence was cloned by RT-PCR in accordance to porcine BPI amino terminal DNA sequence. This cloned sequence was inserted into prokaryotic expression vector pET32a(+). Being induced, recombination protein was separated and purified and assayed its bactericidal activity in vitro. The results showed that porcine BPI recombination protein posses bactericidal action for gram-negative bacteria but no effect for gram-positive bacteria. Its most power full effect was in 20 minutes to 2 hours. Porcine BPI recombination protein still had apparente bactericidal action when it was maintained in-20℃,4℃,20℃,40℃and 60℃for 30 minutes. It had more powerful bactericidal action in acidity and low density condition. Actually, a strain of porcine recombination antiseptic protein genetic engineering bacterium was built. Which was named E. coli BL21(DE3)-pET32a(+)-pBPIN and had been preserved in China Center for Type Culture Collection (CCTCC) with accession No. CCTCC M 2011252.4. Specific primers, which contain specified restriction enzyme cutting site, was designed and its DNA sequence was cloned by RT-PCR in accordance to porcine BPI amino terminal DNA sequence. This cloned sequence was inserted into eukaryotic expression vector pcDNA3.1(+). Which was transfected into Vero cell and its transcription product and recombination protein were detected by RT-PCR and Western blot, respectively. Mice bilateral tibialis anterior muscle were immunized by recombinant plasmid via electrical impulse. The mice were infected with Salmonella typhimurium after four times. Mice body temperature and the number of leukocyte, neutrophilic granulocyte and lymphocyte number were detected after four weeks. T-test was analysed by SPSS software between the control group and recombinant plasmid group. The results showed that there was no significant difference in lymphocyte number and neutrophile granulocyte number between control group and recombinant plasmid group(P>0.05) and significant difference in leucocyte number(P<0.01) in the first week. And significant difference in leucocyte number. lymphocyte number and neutrophile granulocyte number between control group and recombinant plasmid group(P<0.01). The results illustrated that anti-inflammatory ability of injected recombinant plasmid mice was much better than that of in the control group. |
