Studies on molecular cloning of the pgm gene of piscine Streptococcus iniae and recombinant bacteria

• Main Authors: Wang Yu-Chen, 王玉珍
• Other Authors: Lin Cheng-Hui
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/95751997322805073280

碩士 === 國立臺灣海洋大學 === 水產養殖學系 === 95 === Abstract 　　Streptococcus iniae causes a fatal meningoencephalitis and is associated with high mortality in farmed fishes. It may appear chronic or unobvious infection. Characteristic symptoms include exophthalmia (which may be bilateral), corneal opacity, melanosis, lethargy, loss of orientation, swimming erratically, dorsal rigidity, vertebral deformity, tachypnoea, anorexia, emaciation, or petechiae around the anal zone. This study amplified the pgm gene of Streptococcus iniae ATCC 29178 by using polymerase chain reaction (PCR). The PCR product was 762 bps and contained Xho I and Hind III recognition site. DNA sequence of the PCR product was the same as that of the pgm gene of S. iniae ATCC 29178. The PCR product was cloned into pBluescriptⅡto generate pBS-pgm. Then, the pgm DNA fragment was digested with Xho I and Hind III and ligated with pGL3-Basic digested with Xho I and Hind III to generate pGL3-pgm. Kanamycine resistant gene was amplified from pEGFP-N1 by PCR with primer Kan-Pst I F and Kan-Pst I R. The kanamycine resistant gene and pGL3-pgm were digested with Pst I and then ligated to generate pGL3-P-K. The pGL3-P-K was digested with Xho I and Hind III to generate P-K DNA fragment. Allelic replacement of the Streptococcus iniae wild type with the pGL3-P-K and P-K fragment was accomplished by homologous recombination. The recombinant S. iniae P-K was selected with kanamycine medium. S. iniae wild type and S. iniae P-K were intraperitoneally injected into tilapia. The result showed that S. iniae P-K had lower virulence.