Preparation of a recombinant tumor-targeting ribosome inactivating protein luffin-α-NGR and evaluation of its antitumor activity / 生物工程学报

Loofah seeds ribosome inactivating protein luffin-α was fused with a tumor-targeting peptide NGR to create a recombinant protein, and its inhibitory activity on tumor cells and angiogenesis were assessed. luffin-α-NGR fusion gene was obtained by PCR amplification. The fusion gene was ligated with pGEX-6p-1 vector to create a recombinant plasmid pGEX-6p-1/luffin-α-NGR. The plasmid was transformed into E. coli BL21, and the target protein was isolated and purified by GST affinity chromatography. The luffin-α-NGR fusion gene with a full length of 849 bp was successfully obtained, and the optimal soluble expression of the target protein was achieved under the conditions of 16 ℃, 0.5 mmol/L IPTG after 16 h induction. SDS-PAGE and Western blotting confirmed the recombinant protein has an expected molecular weight of 56.6 kDa. Subsequently, the recombinant protein was de-tagged by precision protease digestion. The inhibitory effects of the recombinant protein on liver tumor cells HepG2 and breast cancer cells MDA-MB-231 were significantly stronger than that of luffin-α. The Transwell and CAM experiment proved that the recombinant protein luffin-α-NGR also had a significant inhibitory effect on tumor cells migration and neovascularization. The inhibitory activity on tumor cells and angiogenesis of the recombinant luffin-α-NGR protein lays a foundation for the development of subsequent recombinant tumor-targeting drugs.

Research progress of Phytolaccae Radix / 中草药

This paper summarizes the herbal textual research, authenticity identification, processing, composition and pharmacology, clinical studies, industry and agriculture resources development of Phytolaccae Radix based on the ancient herbology works and references. The herbal textual research showed that Phytolaccae Radix had different names like Shanglu or Danglu or Zhanglu. The effects are external application of carbuncle, swelling and sore toxin, internal administration of diuretic and hydroncus. The main producing area is transferred from northwest to southeast. The root has the abnormal structure of concentric ring, which is different from the eight kinds of common adulterants. It is mainly processed with vinegar to reduce toxicity and increase efficacy. Triterpenoid saponins, polysaccharides and pokeweed antiviral proteins (PAPs) are the main effective components of Phytolaccae Radix, which have the functions of diuresis and diarrhea, improving inflammation of kidney, liver, respiratory tract and neuroinflammatory diseases, as well as antibacterial, antiviral, antitumor and other pharmacological activities. It is mainly used for the treatment of cirrhosis ascites, nephrotic syndrome and external application for the treatment of constipation clinically. Phytolaccae Radix has important application value in plant disease resistance, insect resistance and environmental restoration. The clinical efficacy of Phytolaccae Radix is clear and it has made important progress in the research of components, pharmacology, industrial and agricultural resources development, which provides support for the rational development and comprehensive utilization of it.

Molecular cloning and prokaryotic expression of a type II ribosome inactivating protein from Polyporus umbellatus / 药学学报

A type Ⅱ ribosome inactivating protein (RIP) gene was cloned from Polyporus umbellatus sclerotia by RT-PCR method. The full open reading frame cDNA sequence of this gene was 873 bp in length and encoded a 290-aa protein with a molecular weight of 32.33 kDa and an isoelectric point of 5.58. Multiple sequence alignment revealed that the deduced amino acids possessed conserved domains of RICIN superfamily protein. A neighbor joining phylogenetic analysis suggests that PuRIP was closely related to RIP in Marasmius oreades. Real time PCR results showed that this gene expressed in all tested tissues of P. umbellatus. Meanwhile, the expression of this gene was significantly up-regulated in the part infected by Armillaria mellea. This result suggested that this PuRIP might played important role with potential biotic stress tolerance of P. umbellatus. Otherwise, we successfully constructed the pET15b-PuRIP plasmid, produced and purified the His-PuRIP fusion protein, which would provide the basic material for polyclonal antibody preparation and gene function research.

Ricin and its antidote agents:research advances / 国际药学研究杂志

Ricin is a highly toxic plant protein produced by the seeds of the castor plant. It belongs to the ribosome inactivat-ing proteins(RIP)family and causes cell death by inhibiting the protein synthesis activity of ribosome. Ricin takes a unique pathway calledretrograde trafficking pathwayto enter the cytosol,where it interacts with ribosome and then exerts its inhibitory activity. No effective antidote agents have been developed for the treatment of ricin poisoning. In this paper,the structure,cell trafficking process, toxicological mechanism and the research progress in ricin antitoxin agents are reviewed.

Ricin and its antidote agents: Research advances / 国际药学研究杂志

Ricin is a highly toxic plant protein produced by the seeds of the castor plant. It belongs to the ribosome inactivating proteins (RIP) family and causes cell death by inhibiting the protein synthesis activity of ribosome. Ricin takes a unique pathway called "retrograde trafficking pathway" to enter the cytosol, where it interacts with ribosome and then exerts its inhibitory activity. No effective antidote agents have been developed for the treatment of ricin poisoning. In this paper, the structure, cell trafficking process, toxicological mechanism and the research progress in ricin antitoxin agents are reviewed.

Toxicity and treament of ricin poisoning:a review / 中国药理学与毒理学杂志

Ricin is a plant toxin isolated from the seed of the castor plant(Ricinus communis). As a typical II ribosome inactivating protein,ricin consists of two polypeptide chains named ricin toxin A chain(RTA)and ricin toxin B chain(RTB),linked via a disulfide bridge. RTB binds to both glycopro?tein and glycolipid at the surface of the target cell and mediates ricin to be endocytosed and transported retro?gradely to the endoplasmic reticulum. After being reduced and retrotranslocated to the cytosol,RTA mediates its toxicity due to its activity of a RNA N- glycosidases. Aside from its main toxic effect of protein synthesis inhibition,ricin also displays other properties that contribute to its toxicity such as inducing apoptosis,cytokine secreting and peroxidation. Ricin is stable and can be easily isolated. It has many routes of intoxication with no specific antidotes. Due to its natural abundance,remarkable toxicity,and the potential to be used in biological warfare as well as terrorist attacks,ricin has been classified as a Category B biothreat agent. Here we reviewed its history as a biothreat agent,constitu?tion,intoxication mechanism,detection methods and the development of specific antitodes.

Structural studies on a non-toxic homologue of type II RIPs from bitter gourd: Molecular basis of non-toxicity, conformational selection and glycan structure.

The structures of nine independent crystals of bitter gourd seed lectin (BGSL), a non-toxic homologue of type II RIPs, and its sugar complexes have been determined. The four-chain, two-fold symmetric, protein is made up of two identical two-chain modules, each consisting of a catalytic chain and a lectin chain, connected by a disulphide bridge. The lectin chain is made up of two domains. Each domain carries a carbohydrate binding site in type II RIPs of known structure. BGSL has a sugar binding site only on one domain, thus impairing its interaction at the cell surface. The adenine binding site in the catalytic chain is defective. Thus, defects in sugar binding as well as adenine binding appear to contribute to the non-toxicity of the lectin. The plasticity of the molecule is mainly caused by the presence of two possible well defined conformations of a surface loop in the lectin chain. One of them is chosen in the sugar complexes, in a case of conformational selection, as the chosen conformation facilitates an additional interaction with the sugar, involving an arginyl residue in the loop. The N-glycosylation of the lectin involves a plant-specific glycan while that in toxic type II RIPs of known structure involves a glycan which is animal as well as plant specific.

A Novel Method for Assaying Enzymatic Activity of Ribosome-inactivating Protein Using Plasmid DNA as Substrate / 生物化学与生物物理进展

A novel method for assaying the enzymatic activity of ribosome-inactivating proteins(RIPs) has been developed.The principle of the method is based on that RIP can remove some adenine bases from double-stranded supercoiled DNA molecules,subsequently,the deadenylated DNA was cleaved into nicked and linear form.After treatment with acidic aniline,the deadenylated DNA was degraded into many small fragments,and run out of the gel.The enzymatic activities of two RIPs(trichosanthin and cinnamomin) were tested using this method,the limit of sensitivity is about 50 ng(trichosanthin) and 5 ng(reduced cinnamomin) .It should be emphasized that the merit of this method is to avoid the preparation of ribosome.

Immunotoxins composed of monoclonal antihuman T Iymphocyte antibody and single chain ribosome—inactivating proteins:antitumor effects in vitro and in Vivo / 中国免疫学杂志

Two new single chain ribosome—inactivating proteins,polygonin2 from the roots ofPolygonatum odoratum and momordin2,from the seeds of Momordica charantia,inhinited pro-teib synthesis potentially in rabbit reticulocyte lysate,but were relatively low toxic to Molt—4 cells.polygonin2 and momordin2 were conjugated to H65 monoclonal antibody that recognizedhuman T lymphocyte CD5 surface antigen using a heterobifunctional crosslinking reagent 2—iminothiolane.The resulting immunotoxins,referred to as H65—PGN2 and H65—MOR2,showed potent specific cytotoxicity to target cell Molt—4 and human peripheral blood T lympho-cyte,but had no effect on human hematopoietic cells.Injection of Molt—4 cells s.c.into Balb/cnu/nu mice developed into a solid tumor.Administration of the H65—PGN2 or H65—MOR2immunotoxins i.p.10 ug per mice at 48—h intervals suppressed tumor growth specifically.Theresulting showed that clinical studied were warranted to use these conjugates for the therapy ofautoimmune diseases as well as the ex vivo purging of T lymphocyte in ABMT.