Recombinant Expression Of Anti-lipopolysaccharide Factor (ALFFc) From The Chinese Shrimp

Shrimp aquaculture has increased rapidly from an insignificant base in the early 1980s, it has brought considerable economic benefits for coastal areas. But the shrimp diseases also attack the shrimp aquaculture and economy. In recent years, shrimp immunological study has been progressed rapidly. The immune-related genes which have been cloned continuously were studied deeply. Antilipopolysaccharide factor (ALF) is an antimicrobial peptid found in the horseshoe crab and crustacean animals.ALFs have a broad range of antimicrobial activity. The deep research about ALF will benefit to the control of shrimp diseases. In this paper, the recombinant expression of ALF of Fenneropenaeus chinensis (ALFFc) was studied, including the following four aspects.1 The primers was designed according to ALFFc gene, a prokaryotic expression vector pET-DsbA-ALFFc was constructed and expressed in Escherichia. coli (DE3). Because of the expression product of ALFFc gene killing itself host by conventional inducing, it's difficult to obtain recombinant protein. So we tried to find three induction tactics including low"temperature method","DTT induced method"and"IPTG screening method"to achieve high expression of recombinant protein.2 A large number of proteins were induced by IPTG screening method which was the best induction method by comparing of the three induction methods. The purified recombinant protein DsbA-ALFFc was purified by His affinity column and was.used as antigen to prepare antiserum in New Zealand white rabbit. We got a specific band in the expected position by Western Blot detection.3 A eukaryotic expression vector pET-DsbA-ALFFc was constructed and expressed successfully in GS115 host, and we also explored the fermentation conditions. Using the antiserum prepared above, we did a Western Blot detect of the eukaryotic expression product, and finally we got a specific band in the expected position, this showed that we had expressed the target protein successfully.4 The same amount of shrimp tissue protein samples of hemocyte, gill, intestine, ovary, hepatopancreas, serum and muscle was separated by 15% SDS electrophoresis, and then were identified by Western Blot. The results showed that the ALFFc existed mainly in hemocyte, there were low specific signals in gill.and no signals were detected in serum and other organizations. These results indicated that ALFFc storaged mainly in hemocyte of uninfected shrimp.