Fenfang Wu

Lamprey Variable Lymphocyte Receptors Mediate Complement-Dependent Cytotoxicity

An alternative adaptive-immune system is present in the most basal vertebrates—lampreys and hagfish—the only surviving jawless vertebrates. These eel-like fish use leucine-rich repeat–based receptors for Ag recognition instead of the Ig-based receptors used in jawed vertebrates. We report that in Japanese lamprey (Lampetra japonica), variable lymphocyte receptor (VLR)B interacts with C1q and C3 proteins to mediate complement-dependent cytotoxicity for bacteria and tumor cells. The immune-based lysis involves deposition of VLRB and C1q-like protein complex on the surface of target cells, activation of C3, and ultimate disruption of cell wall integrity. The demonstration of functional interaction between VLRB and complement components in lamprey provides evidence for the emergence of cooperative innate and adaptive-immune responses at a pivotal point in vertebrate evolution, before or in parallel with the evolution of Ig-based Abs and the classical complement-activation pathway.

Cloning of Arctic lamprey Lethenteron camtschaticum cd9 with roles in the immune response

In this study, the cd9 gene, a member of the tetraspanin superfamily and involved in various cellular processes, was cloned from Lethenteron camtschaticum. Both real-time PCR and immunohistochemical assays showed broad distribution of cd9 in various L. camtschaticum tissues. In addition, expression levels of Cd9 mRNA were up-regulated in the liver and heart after stimulation by lipopolysaccharide. Flow cytometric analyses demonstrated that cd9 was detected on the leukocytes and that the expression level was higher on granulocytes than on lymphocytes, which implied that cd9 was mainly involved in innate immunity.

Effects of the recombinant toxin protein rLj-RGD3 in multidrug-resistant human breast carcinoma cells.

We have previously reported that Lj-RGD3, a novel RGD-toxin protein, was isolated from the buccal gland of Lampetra japonica. The recombinant protein rLj-RGD3 has anti-invasive and anti-adhesive activity in tumor cells (HeLa cells) and endothelial cells (ECV304 cells) in vitro, and inhibits αvβ3, αvβ5, and β1 integrin-mediated adhesion. In this study, we investigated the bioactivity of rLj-RGD3 in the drug-resistant MCF-7/Adr breast carcinoma cell line and drug-sensitive parental line MCF-7, and found that rLj-RGD3 inhibited the growth of both cell lines. Biological function studies revealed that rLj-RGD3 could induce the apoptosis in MCF-7/Adr, which was more prevalent than that in the drug-sensitive parental line MCF-7. In addition, rLj-RGD3 inhibited the adhesion of MCF-7/Adr cells to fibronectin. Furthermore, rLj-RGD3 prevented invasion of MCF-7/Adr cells through an artificial matrigel basement membrane. In summary, rLj-RGD3 may be used as a potential drug in multidrug-resistant breast cancer therapy.

Detection of the Single-Chain Precursor in the Production and Purification Process of Recombinant Human Insulin

High quality recombinant insulin requires being free of single-chain precursor (proinsulin), a task that depends on the selectivity and sensitivity of the monitoring process for detecting proinsulin. In this study we developed an enzyme-linked immunosorbent assay (ELISA) system that was specifically tailored to detect recombinant proinsulin. The proinsulin consists of six components: an initiating methionine, 48 amino acids from human growth hormones (HGH, used as the protection peptide), first connecting Arg-residue, B-chain of insulin, and second connecting Arg-peptide and A-chain of insulin. This form of proinsulin is more stable and can be efficiently expressed by E. coli than insulin. Herein, we evaluated the specificity, precision, recovery, sensitivity, and detection range of the proinsulin ELISA kit. The results showed that the ELISA kit is a very useful tool for monitoring the proinsulin yield in early stages of insulin production as well as the residual proinsulin in the final product, insulin.

Monoclonal and polyclonal antibodies against the precursor of recombinant human insulin

The precursor of recombinant human insulin is also called recombinant proinsulin. It is a fusion protein containing protection peptide and insulin. The protection peptide contains 55 amino acids (48 amino acids from human growth hormone, and a connecting peptide containing Arg-residues) and can effectively improve protein stability and expression efficiency [1]. However, in clinical trials the presence of proinsulin in the insulin final products was found to have a negative effect on the heart, leading to an increased incidence of myocardial infarction [2–6]. More importantly, recombinant proinsulin is a heterologous protein. Therefore, the removal of the recombinant proinsulin in the final product of insulin is vital, and the effectiveness of its removal should be analyzed by appropriate methods. In this study, we prepared specific monoclonal and polyclonal antibodies against proinsulin and established enzyme-linked immunosorbent assay (ELISA) for the detection of the residual precursor in the insulin production. Three adult female BALB/c mice were intraperitoneally injected with 55-peptide as an antigen. Each mouse was injected with 100 mg antigen mixed with complete Freund’s adjuvant (Sigma, St Louis, USA) the first time, and then boosted with 50 mg antigen mixed with incomplete Freund’s adjuvant for three times. The injection interval was 2 weeks. After four immunizations, spleen cells were fused with SP2/0 myeloma cells and the hybridoma cells were selected by growing in HAT (hypoxanthine, aminopterin, and thymidine) medium. Monoclonal cell lines were screened by the limiting dilution method. To obtain a large amount of antibodies, the hybridoma cells were injected into the peritoneal cavity of mice, and antibody-rich ascites was collected. The antibodies were purified by protein-G Sepharose (GE Healthcare, Wisconsin, USA). The titer of anti-55-peptide monoclonal antibody (mAb) was determined by indirect ELISA. New Zealand white rabbits were immunized with the purified recombinant proinsulin (but not insulin). If the animal is immunized with pure insulin, it would soon become sick and die. Each rabbit was hypodermically injected with 600 mg antigen mixed with complete Freund’s adjuvant the first time, and then boosted three times with 300 mg antigen mixed with incomplete Freund’s adjuvant. The injection interval was 2 weeks. Seven days after the last immunization, the serum of the immunized animals was collected. The anti-insulin polyclonal antibodies (pAbs) in the antiserum were purified by affinity chromatography using recombinant insulin (purchased from Novo Nordisk, Bagsvaerd, Denmark) coupled CNBr-activated Sepharose (GE Healthcare). The titer of the anti-insulin pAbs was assayed by indirect ELISA. Microtiter plates (Coring, New York, USA) were coated with 20 mg anti-insulin pAbs, and then blocked with 1% bovine serum albumin (BSA) overnight at 48C. The samples (proinsulin, insulin, and 55-peptide) were added to each well, respectively. The plate was incubated for 90 min at 378C and washed three times with PBST (10 mM PBS þ 0.05% Tween-20). Anti-55-peptide mAbs were added and incubated for 90 min at 378C. The plate was washed three times with PBST and then incubated with horse radish peroxidase (HRP)-conjugated goat anti-mouse IgG for 90 min at 378C. After washing, tetramethylbenzidine solution was added into wells and incubated for 10–15 min. Finally, 100 ml of 2 M H2SO4 solution was added to terminate the reaction. The absorbance was read at dual filter 450/620 nm. The anti-insulin pAbs were efficiently purified with affinity chromatography, and the antibody heavy chains and light chains could be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [Fig. 1(A)]. The titer for the anti-insulin pAbs was 1 : 32,000 as determined by indirect ELISA [Fig. 1(C)]. The anti-55-peptide mAbs were purified by protein-G Sepharose and both the heavy and light chains of the antibody were detected by SDS-PAGE [Fig. 1(B)]. The titer was 1 : 32,000 [Fig. 1(D)]. Acta Biochim Biophys Sin 2013, 45: 704–705 | a The Author 2013. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmt041. Advance Access Publication 23 April 2013

Preparation method of lamprey antisera and activity assay

Lampreys, the surviving representative of jawless vertebrates, have been a focal point in the search for the evolutionary origin of adaptive immunity. They have independently evolved the variable lymphocyte receptor (VLR)-based adaptive immune system that protects themselves from infection by a variable of microorganisms. The standard immunization schedule for Japanese lamprey (Lampetra japonica) was established to prepare antisera by injection of Escherichia coli, Bacillus proteus, Staphylococcus aureus, Mycobacterium smegmatis, RRBCs, SRBCs, NB4 cells and Hela cells. In this study, we demonstrated the activities of lamprey antisera, which might be helpful to research the collaboration between VLR-based adaptive immune system and complement system in jawless vertebrates.