Molecular cloning, characterization, and expression analysis of two different types of lectins from the oriental river prawn, Macrobrachium nipponense.

Lectins, which are widely expressed in invertebrates, play important roles in many biological processes, including protein trafficking, cell signaling, pathogen recognition, as effector molecules, and so on (Wang and Wang, 2013). This study identified one novel M-type lectin and one L-Type lectin, designated as MnMTL1 and MnLTL1, from the oriental river prawn Macrobrachium nipponense. The full-length cDNA of MnMTL1 was 2064 bp with a 1761 bp ORF encoding a putative protein of 586 deduced amino acids. The full-length cDNA of MnLTL1 was 1744 bp with a 972 bp ORF encoding a 323-amino acid peptide. The deduced MnMTL1 protein contained a putative type II transmembrane region and a 440-aa Glycoside hydrolase family 47 (GH47) domain. One luminal carbohydrate recognition domain and a 23-aa type I transmembrane region were identified from the MnLTL1. MnMTL1 shared 78% identity with Marsupenaeus japonicus M-type lectin and MnLTL1 shared 83% similarity with M. japonicus L-type lectin. RT-PCR analysis showed that MnMTL1 and MnLTL1 were expressed in all tested tissues. Quantitative real-time PCR analysis revealed that MnMTL1 and MnLTL1 are substantially fluctuant during Aeromonas hydrophila and Aeromonas veronii infections. Based on immune responses and previous literature, we assumed that MnMTL1 and MnLTL1 might be functioned as pattern recognition receptors and play important roles in the immune response of M. nipponense.

[An expression T-vector and its application at low temperatures].

In modern biology and biotechnology research, recombinant gene expression has been the most popular method to obtain the target protein. In recent years, many foreign genes have been efficiently expressed in Escherichia coli. However, proteins encoded by animal, plant or mesophilic microbial genes often lose activities or become denatured within a few hours at regular growth temperatures for E. coli; some other target proteins are toxic to host cells and therefore difficult to be over-expressed. The new T-vector, pEXC-T, was constructed by combining TA cloning and cold-shock induction to obtain high expression levels with low costs. This paper reports the construction of pEXC-T and optimization of induction techniques for gene expression. Two instable proteins were tested and successfully expressed in soluble form by using pEXC vector. The development of pEXC-T offers a convenient technique for the preparations of recombinant proteins to be used in structure/function studies, or as diagnostic markers and medicinal proteins.