Multiplex Profiling of Biomarker and Drug Uptake in Single Cells Using Microfluidic Flow Cytometry and Mass Spectrometry.

To perform multiplex profiling of single cells and eliminate the risk of potential sample loss caused by centrifugation, we developed a microfluidic flow cytometry and mass spectrometry system (µCytoMS) to evaluate the drug uptake and induced protein expression at the single cell level. It involves a microfluidic chip for the alignment and purification of single cells followed by detection with laser-induced fluorescence (LIF) and inductively coupled plasma mass spectrometry (ICP-MS). Biofunctionalized nanoprobes (BioNPs), conjugating ∼3000 6-FAM-Sgc8 aptamers on a single gold nanoparticle (AuNP) (Kd = 0.23 nM), were engineered to selectively bind with protein tyrosine kinase 7 (PTK7) on target cells. PTK7 expression induced by oxaliplatin (OXA) uptake was assayed with LIF, while ICP-MS measurement of 195Pt revealed OXA uptake of the drug in individual cells, which provided further in-depth information about the drug in relation to PTK7 expression. At an ultralow flow of ∼0.043 dyn/cm2 (20 µL/min), the chip facilitates the extremely fast focusing of BioNPs labeled single cells without the need for centrifugal purification. It ensures multiplex profiling of single cells at a throughput speed of 500 cells/min as compared to 40 cells/min in previous studies. Using a machine learning algorithm to initially profile drug uptake and marker expression in tumor cell lines, µCytoMS was able to perform in situ profiling of the PTK7 response to the OXA at single-cell resolution for tests done on clinical samples from 10 breast cancer patients. It offers great potential for multiplex single-cell phenotypic analysis and clinical diagnosis.

Tracking and imaging nano-plastics in fresh plant using cryogenic laser ablation inductively coupled plasma mass spectrometry.

Tracking and imaging of nano-plastics are extremely challenging, especially in fresh biological samples. Here, we propose a new strategy in which polystyrene (PS) was doped with the europium chelate Eu (DBM)3bpy to quantify, track, and in situ image nano-plastics in fresh cucumber based on inherent metals using cryogenic laser ablation inductively coupled plasma mass spectrometry (cryo-LA-ICP-MS). The cryogenic conditions provide a stable condition for imaging fresh cucumber, suppressing the evaporation of water in fresh plants, and maintaining the original structure of plants with respect to room temperature imaging in LA-ICP-MS. The plants were cultivated in two types of nano-plastics solutions with low (50 mg/L) and high (200 mg/L) concentrations for 9 days. The results showed that nano-plastics mainly enrich the roots and have negative effects, which decrease the trace elements of Zn, Mn, and Cu in cucumber. Smaller PS particles are able to penetrate the plant more easily and inflict serious damage. Novel imaging method provides a novel insight into the tracking and imaging of nano-plastics in fresh plant samples. The results illustrated that nano-plastics deposition on plants has the potential to have direct ecological effects as well as consequences for potential health.

Dean-Flow-Coupled Elasto-Inertial Focusing Accelerates Exosome Purification to Facilitate Single Vesicle Profiling.

Exosomes are recognized as noteworthy biomarkers playing unprecedented roles in intercellular communication and disease diagnosis and treatment. It is a prerequisite to obtain high-purity exosomes for the comprehension of exosome biochemistry and further illustration of their functionality/mechanisms. However, the isolation of nanoscale exosomes from endogenous proteins is particularly challenging for small-volume biological samples. Herein, a Dean-flow-coupled elasto-inertial microfluidic chip (DEIC) was developed. It consists of a spiral microchannel with dimensional confined concave structures and facilitates elasto-inertial separation of exosomes with lower protein contaminants from cell culture medium and human serum. The presence of 0.15% (w/v) poly-(oxyethylene) controls the elastic lift force acting on suspended nanoscale particles and makes it feasible for field-free purification of integrity exosomes with a 70.6% recovery and a 91.4% removal rate for proteins. As a proof of concept, the technique demonstrated the individual-vesicle-level biomarker (EpCAM and PD-L1) profiling in combination with simultaneous aptamer-mediated analysis to disclose the sensibility for immune response. Overall, DEIC enables the collection of high-purity exosomes and exhibits potential in integration with downstream analyses of exosomes.

Intolerance of profligacy: an aptamer concentration gradient-tailored unicellular array for high-throughput biologics-mediated phenotyping.

In aptamer-based assay schemes, aptamer probes not labeled with biomarkers have to be eliminated before testing, which may lead to a tremendous waste of precious probes. We herein propose a microfluidics system integrating an aptamer concentration gradient generator (Apt-CGG) and a dual single-cell culturing array (D-SCA), termed Mi-Apt-SCA. This facilitates the precise construction of a nanoscale-gradient microenvironment and the high-throughput profiling of single-cell growth/phenotypes in situ with the minimal consumption of Apt-probe. Unlike previous snakelike mixers, the choreographed winding-ravined aptamer dual-spiral micromixer (Apt-WD-mixer) in Apt-CGG could allow thorough blending to generate linear concentration gradients of aptamer (quasi-non-Newtonian fluid) under the action of continuous fluidic wiggles and bidirectional Dean flow. In contrast to other trap-like systems, the mild vortex allows single-cell growth in an ultra-tender fluidic microenvironment using triple-jarless single-cell culture capsules (TriJ-SCCs) in D-SCA (shear stress: 3.43 × 10-5 dynes per cm2). The minimum dosage of aptamer probe required for exploring PDL1 protein expression in two hepatoma cell lines is only one-900th of that required by conventional protocols. In addition, this approach facilitated the profiling of ITF-ß/cisplatin-mediated single-cell/cell-cluster phenotypes.

Acinar ATP8b1/LPC pathway promotes macrophage efferocytosis and clearance of inflammation during chronic pancreatitis development.

Noninflammatory clearance of dying cells by professional phagocytes, termed efferocytosis, is fundamental in both homeostasis and inflammatory fibrosis disease but has not been confirmed to occur in chronic pancreatitis (CP). Here, we investigated whether efferocytosis constitutes a novel regulatory target in CP and its mechanisms. PRSS1 transgenic (PRSS1Tg) mice were treated with caerulein to mimic CP development. Phospholipid metabolite profiling and epigenetic assays were performed with PRSS1Tg CP models. The potential functions of Atp8b1 in CP model were clarified using Atp8b1-overexpressing adeno-associated virus, immunofluorescence, enzyme-linked immunosorbent assay(ELISA), and lipid metabolomic approaches. ATAC-seq combined with RNA-seq was then used to identify transcription factors binding to the Atp8b1 promoter, and ChIP-qPCR and luciferase assays were used to confirm that the identified transcription factor bound to the Atp8b1 promoter, and to identify the specific binding site. Flow cytometry was performed to analyze the proportion of pancreatic macrophages. Decreased efferocytosis with aggravated inflammation was identified in CP. The lysophosphatidylcholine (LPC) pathway was the most obviously dysregulated phospholipid pathway, and LPC and Atp8b1 expression gradually decreased during CP development. H3K27me3 ChIP-seq showed that increased Atp8b1 promoter methylation led to transcriptional inhibition. Atp8b1 complementation substantially increased the LPC concentration and improved CP outcomes. Bhlha15 was identified as a transcription factor that binds to the Atp8b1 promoter and regulates phospholipid metabolism. Our study indicates that the acinar Atp8b1/LPC pathway acts as an important "find-me" signal for macrophages and plays a protective role in CP, with Atp8b1 transcription promoted by the acinar cell-specific transcription factor Bhlha15. Bhlha15, Atp8b1, and LPC could be clinically translated into valuable therapeutic targets to overcome the limitations of current CP therapies.

Dual-Multivalent-Aptamer-Conjugated Nanoprobes for Superefficient Discerning of Single Circulating Tumor Cells in a Microfluidic Chip with Inductively Coupled Plasma Mass Spectrometry Detection.

The efficient recognition of circulating tumor cells (CTCs) with an aptamer probe confers numerous benefits; however, the stability and binding affinity of aptamers are significantly hampered in real biological sample matrices. Inspired by the efficient preying mechanism by multiplex tubing feet and endoskeletons of sea urchins, we engineered a superefficient biomimetic single-CTC recognition platform by conjugating dual-multivalent-aptamers (DMAs) Sgc8 and SYL3C onto AuNPs to form a sea urchin-like nanoprobe (sea urchin-DMA-AuNPs). Aptamers Sgc8 and SYL3C selectively bind with the biomarker proteins PTK7 and EpCAM expressed on the surface of CTCs. CTCs were captured with 100% efficiency, followed by sorting on a specially designed multifunctional microfluidic configuration, integrating a single-CTC separation unit and a hydrodynamic filtrating purification unit. After sorting, background-free analysis of biomarker proteins in single CTCs was undertaken with inductively coupled plasma mass spectrometry by measuring the amount of 197Au isotope in sea urchin-DMA-AuNPs. With respect to a single-aptamer nanoprobe/-interface, the dual-aptamer nanoprobe improves the binding efficiency by more than 200% (Kd < 0.35 nM). The microchip facilitates the recognition of single CTCs with a sorting separation rate of 93.6% at a flow rate of 60 µL min-1, and it exhibits 73.8 ± 5.0% measurement efficiency for single CTCs. The present strategy ensures the manipulation and detection of a single CTC in 100 µL of whole blood within 1 h.

Peroral traction-assisted natural orifice trans-anal flexible endoscopic rectosigmoidectomy followed by intracorporeal colorectal anastomosis in a live porcine model.

BACKGROUND: Compared to traditional open surgery, laparoscopic surgery has become a standard approach for colorectal cancer due to its great superiorities including less postoperative pain, a shorter hospital stay, and better quality of life. In 2007, Whiteford et al reported the first natural orifice trans-anal endoscopic surgery (NOTES) sigmoidectomy using transanal endoscopic microsurgery. To date, all cases of NOTES colorectal resection have included a hybrid laparoscopic approach with the use of established rigid platforms. AIM: To introduce a novel technique of peroral external traction-assisted transanal NOTES rectosigmoidectomy followed by intracorporeal colorectal end-to-end anastomosis by using only currently available and flexible endoscopic instrumentation in a live porcine model. METHODS: Three female pigs weighing 25-30 kg underwent NOTES rectosigmoid resection. After preoperative work-up and bowel preparation, general anesthesia combined with endotracheal intubation was achieved. One dual-channel therapeutic endoscope was used. Carbon dioxide insufflation was performed during the operation. The procedure of trans-anal NOTES rectosigmoidectomy included the following eight steps: (1) The rectosigmoid colon was tattooed with India ink by submucosal injection; (2) Creation of gastrostomy by directed submucosal tunneling; (3) Peroral external traction using endoloop ligation; (4) Creation of rectostomy on the anterior rectal wall by directed 3 cm submucosal tunneling; (5) Peroral external traction-assisted dissection of the left side of the colon; (6) Trans-anal rectosigmoid specimen transection, where an anvil was inserted into the proximal segment after purse-string suturing; (7) Intracorporeal colorectal end-to-end anastomosis using a circular stapler by a single stapling technique; and (8) Closure of gastrostomy using endoscopic clips. All animals were euthanized immediately after the procedure, abdominal exploration was performed, and the air-under-water leak test was carried out. RESULTS: The procedure was completed in all three animals, with the operation time ranging from 193 min to 259 min. Neither major intraoperative complications nor hemodynamic instability occurred during the operation. The length of the resected specimen ranged from 7 cm to 13 cm. With the assistance of a trans-umbilical rigid grasper, intracorporeal colorectal, tension-free, end-to-end anastomosis was achieved in the three animals. CONCLUSION: Peroral traction-assisted transanal NOTES rectosigmoidectomy followed by intracorporeal colorectal end-to-end anastomosis is technically feasible and reproducible in an animal model and is worthy of further improvements.

Treatment With Liraglutide Exerts Neuroprotection After Hypoxic-Ischemic Brain Injury in Neonatal Rats via the PI3K/AKT/GSK3ß Pathway.

Neonatal hypoxic-ischemic (HI) brain injury is a detrimental disease, which results in high mortality and long-term neurological deficits. Nevertheless, the treatment options for this disease are limited. Thus, the aim of the present study was to assess the role of liraglutide in neonatal HI brain injury in rats and investigate the associated mechanisms. The results showed that treatment with liraglutide significantly reduced infarct volume and ameliorated cerebral edema, decreased inflammatory response, promoted the recovery of tissue structure, and improved prognosis following HI brain injury. Moreover, treatment with liraglutide inhibited apoptosis and promoted neuronal survival both in the rat model and following oxygen-glucose deprivation (OGD) insult. LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K), partially reversed these therapeutic effects, suggesting that the PI3K/protein kinase B (Akt) pathway was involved. In conclusion, our data revealed that treatment with liraglutide exerts neuroprotection after neonatal HI brain injury via the PI3K/Akt/glycogen synthase kinase-3ß (GSK3ß) pathway and may be a promising therapy for this disease.

Characterization of the complete mitochondrial genome of Pelteobagrus intermedius (Nichols et Pope).

Pelteobagrus intermedius is a unique freshwater fish native species in the Hainan Island and Guangxi and other water systems of China. In this study, the complete mitochondrial genome sequence of P. intermedius was determined from whole genome Illumina sequencing data. The total length of the mtDNA was 16,532 bp, including 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a non-coding control region. Phylogenetic analysis suggested that the P. intermedius is closely related to Tachysurus fulvidraco. The results will serve as a helpful reference for further studies on the conservation genetics of genus Pelteobagrus.

Complete mitochondrial genome of the green sunfish (Lepomis cyanellus).

The green sunfish (Lepomis cyanellus) is a species of freshwater fish in the Centrarchidae family of order Perciformes, which is native to a wide area of North America. This species was introduced into China in 1998 and became one of the economically important freshwater species. In this study, we determined the complete mitochondrial genome sequence of L. cyanellus. The total length of L. cyanellus mitogenome is 16,485 bp, which consists of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 control region (D-loop), with the genome organization and gene order being identical to that of the typical vertebrate. The overall base compositions of the heavy strand are 27.5% A, 26.2% T, 29.9% C, and 16.4% G, with an A + T content of 53.7%. The mitochondrial genome of L. cyanellus shared 86.8% sequence identity with that of Lepomis macrochirus. The sequence information presented here will be useful for evolutionary as well as population genetic studied.

Transcriptome profiling and digital gene expression analysis of Nile tilapia (Oreochromis niloticus) infected by Streptococcus agalactiae.

Tilapia is an important freshwater aquaculture species worldwide. In recent years, streptococcal diseases have severely threatened development of tilapia aquaculture, while effective prevention and control methods have not yet been established. In order to understand the immunological response of tilapia to infection by Streptococcus agalactiae (S. agalactiae), this study employed Solexa/Illumina RNA-seq and digital gene expression (DGE) technology to investigate changes in the tilapia transcriptome before and after S. agalactiae infection. We obtained 82,799 unigenes (mean size: 618 bp) using de novo assembly. Unigenes were annotated by comparing against databases including Nr, Swissprot, cluster of orthologous groups of proteins, Kyoto encyclopedia of genes and genomes, and gene ontology. Combined with DGE technology, transcriptomic changes in tilapia before and after bacteria challenging were examined. A total of 774 significantly up-regulated and 625 significantly down-regulated unigenes were identified, among which 293 were mapped to 181 signaling pathways including 17 immune-related pathways involving 65 differentially expressed genes. We observed a change in the expression of six genes in the Toll-like receptor signaling pathway, and this was subsequently confirmed via quantitative real-time PCR. This comparative study of the tilapia transcriptome before and after S. agalactiae infection identified important differentially-expressed immune-related genes and signaling pathways that will provide useful insights for further analysis of the mechanisms of tilapia defense against S. agalactiae infection.

The complete mitochondrial genomes of largemouth bass of the northern subspecies (Micropterus salmoides salmoides) and Florida subspecies (Micropterus salmoides floridanus) and their applications in the identification of largemouth bass species.

The largemouth bass belongs to the family Centrarchidae, which includes two subspecies: the northern subspecies, Micropterus salmoides salmoides, and the Florida subspecies, Micropterus salmoides floridanus. In this study, the complete mitochondrial genomes of the two subspecies were sequenced, and their genetic differences were identified. The mitogenomes of M. s. salmoides and M. s. floridanus are 16,486 and 16,479 bp in length, respectively. The two subspecies consisted of 37 genes (13 protein-coding genes, 2 ribosomal RNA, and 22 transfer RNA), which are typical for vertebrate mtDNA. Phylogenetic analysis provided statistical support for the monophyly of the family Centrarchidae. Comparison of the two subspecies' mitogenomes revealed a relatively high number (450) of single nucleotide polymorphisms (SNPs) in protein-coding genes. We characterized SNPs in the partial cytochrome c oxidase subunit 1 gene of different individuals from three cultured populations, one wild northern subspecies population, and one wild Florida subspecies population. Twenty-eight SNPs were fixed with alternative nucleotides in the two subspecies, which could be used for differentiating them. Based on this gene, phylogenetic tree and genetic distance analyses supported that cultured largemouth bass in China belongs to the northern subspecies.

Identification and expression analysis of three c-type lysozymes in Oreochromis aureus.

Lysozyme is an important molecule of innate immune system for the defense against bacterial infections. Three genes encoding chicken-type (c-type) lysozymes, C1-, C2-, C3-type, were obtained from tilapia Oreochromis aureus by RT-PCR and the RACE method. Catalytic and other conserved structure residues required for functionality were identified. The amino acid sequence identities between C1- and C2-type, C1- and C3-type, C2- and C3-type were 67.8%, 65.7% and 63.9%, respectively. Phylogenetic tree analyze indicated the three genes were firstly grouped to those of higher teleosteans, Pleuronectiformes and Tetraodontiformes fishes, and then clustered to those of lower teleosteans, Cypriniformes fishes. Bioinformatic analysis of mature peptide showed that the three genes possess typical sequence characteristics, secondary and tertiary structure of c-type lysozymes. The three tilapia c-type lysozymes mRNAs were mainly expressed in liver and muscle, and C1-type lysozyme also highly expressed in intestine. C1-type lysozyme mRNA was weakly expressed in stomach, C2- and C3-type mRNAs were weakly expressed in intestine. After bacterial challenge, up-regulation was obvious in kidney and spleen for C1-type lysozyme mRNA, while for C2- and C3-type lysozyme obvious increase were observed in stomach and liver, suggesting that C1-type lysozyme may mainly play roles in defense, while C2- and C3-type lysozyme mainly conduct digestive function against bacteria infection. All the three c-type recombinant lysozymes displayed lytic activity against Gram-negative and Gram-positive bacteria. These results indicated that three c-type lysozymes play important roles in the defense of O. aureus against bacteria infections.

Mitochondrial genome sequence of the bluegill sunfish (Lepomis macrochirus).

The bluegill sunfish (Lepomis macrochirus) belongs to Lepomis genera of the family Centrarchidae, which is an economically important freshwater species in China. This study presents the complete mitochondrial genome of L. macrochirus, which is the first complete sequence from sunfish species. L. macrochirus mitochondrial DNA is 16,489 bp long, with the genome organization and gene order being identical to that of the typical vertebrate.

Isolation of Tanichthys albonubes beta actin gene and production of transgenic Tanichthys albonubes.

A beta actin cDNA of Tanichthys albonubes was isolated through the RT-PCR and RACE approach. The cDNA was 1,787-bp in length, including a 1,128-bp CDS, a 95-bp 5'UTR and a 564-bp 3'UTR. Genomic DNA containing the transcription region and 5'-flanking region was cloned based on the beta actin cDNA by Genome walker. A 3,000-bp beta actin gene promoter was then produced by PCR according to the sequences of the 5'-flanking region and the first intron. This promoter consisted of a 1,800-bp 5'-flanking region, and a 1,200-bp 5'-UTR. 3 transcription elements, CAAT box, CArG motif and TATA box were found in the 5'-flanking region. This promoter was inserted into the vector pDsRed2-1 and microinjected into fertilized eggs of Tanichthys albonubes to prove its transcription activity. The beta actin promoter and GH CDS of Tanichthys albonubes were then fused to construct an expression vector pTLA-GH. GH-transgenic Tanichthys albonubes was obtained by microinjection of the pTLA-GH into the fertilized eggs. Fast-growth individuals were observed in the transgenic group and the body weight of the largest individual was 2.1-fold that of the maximum in its non-transgenic siblings in 100 dph. In addition, a co-injection strategy was employed with pTLA-DsRed and pTLA-GH vector and proven to enhance the efficiency of GH-transgenic fish detection.

[Identification of microsatellite markers associated with growth traits in largemouth bass (Micropterus salmoides L.)].

Forty microsatellite loci were selected to amplify the genomic DNA of cultured stock of largemouth bass (Micropterus salmoides L.) in China. Genotypic differences of these microsatellites loci were analyzed using 2-test between the maximal weight group and minimal weight group. Sixteen microsatellites showing significantly difference (P<0.1) were used in genotyping 121 individuals, and the associations between their genotypes and growth traits were examined. Microsatellite loci of JZL60, JZL67, JZL72, JZL124, MiSaTPW76, MiSaTPW117 and MiSaTPW173 were significantly associated with body weight, body length, and body height (P < 0.05 or P < 0.01). The most favorable genotypes for growth traits were AA at JZL60, BB at JZL67, AC at JZL72, BB at MiSaTPW76 and BC at MiSaTPW117. In addition, a total of 47 alleles for the 16 loci were detected (2 approximately 5 alleles for each locus). The average effective number of alleles (Ae), observed heterozygosity (Ho), expected heterozygosity (He) and mean polymorphic information content (PIC) was 2.938, 0.515, 0.500 and 0.445, respectively, both of which indicated that the population genetic diversity was medium.

The complete mitochondrial genome of the RR-B strain of swordtail (Xiphophorus helleri).

BACKGROUND AND AIMS: The swordtail (Xiphophorus helleri) has been used in studies of behavior, phylogenetics, genetics, physiology, cell biology, cancer research, and biomedicine. Mitochondrial DNA (mtDNA) studies are of pressing need in order to assess the population history of the species. MATERIALS AND METHOD: In this study, we present the complete mtDNA genome sequence of two specimens: one from a RR-B strain, a 27-generation inbred line; and one non-selective swordtail with red eyes and red body color from the Guangzhou market, measuring 16,638 and 16,635 bp, respectively. RESULTS: The genome comprises 13 protein-coding genes, 22 tRNAs, two rRNAs and a major non-coding region. The comparison of the two specimens' mitogenomes revealed a relatively low number (57) of single nucleotide polymorphisms-29 located in protein-coding genes, 11 in rRNA genes, six in tRNA genes, and six in the non-coding region. CONCLUSION: We present an important genetic resource for the RR-B strain of swordtails and swordtail species in general.

Cloning and characterization of the tiger shrimp lysozyme.

Lysozymes are key proteins to invertebrates in the innate immune responses against bacterial infections. A lysozyme gene isolated from tiger shrimp, Penaeus monodon, was cloned, sequenced and characterized. The cDNA consists of a signal peptide of 18 amino acids and a mature peptide of 140 amino acids. The lysozyme is presumed to be a chicken-type lysozyme for it possesses two catalytic sites and eight cysteine residues which are highly conserved across species of chicken-type lysozymes. The lysozyme cDNAs of Penaeus semisulcatus, Litopenaeus vannamei, Macrobrachium nipponense and Macrobrachium rosenbergii were also cloned. High similarities existed among shrimp and prawn lysozymes but phylogenetic relationship of shrimps and prawns based on lysozyme molecules did not quite consistent with traditional taxonomic classification. High mRNA expression was detected in hepatopancreas, haemocytes and gill of tiger shrimp. Recombinant lysozyme exhibited potent lytic activities against fish pathogens providing evidence of the involvement of lysozyme in shrimp immunity.

[Expression of Chinese sturgeon cystatin in yeast Pichia pastoris and its proteinase inhibitory activity analysis].

Cystatin, which widely distributed in both tissues and body fluids of animal and plant, was a superfamily of cysteine proteinase inhibitors. It could form activity-inhibitor complexes with cysteine proteinases to inhibit the hydrolytic activity of proteinases. Cystatin played important roles not only in the inhibition of the proteolytic degradation of fish muscle, but also in biological defense systems against invaders. To explore the functions of fish cystatin and the potential values in fish disease prevention and cure, as well as seafood processing, the recombinant yeast strains which could express Chinese sturgeon cystatin were constructed. First, the cystatin cDNA of Chinese sturgeon, which had been PCR modified, was subcloned into yeast integrated vector pPICZaA. After extracted and purified, the recombinant plasmids were linearized by Sac I. The yeast Pichia pastoris GS115 strain was transformed by use of the Lithium Chloride transformation method, and the recombinant cystatin yeast strains got. After 0.5% methanol induction, SDS-PAGE analysis of the culture supernatant indicated that the yield of recombinant cystatin was about 215mg x L(-1) with the percentage about 73.6%. The recombinant cystatin was purified through Q-Sepharose anion-exchange chromatography, and the purity reached about 94.2%. The inhibitory activity of recombinant cystatin was measured by inhibiting the proteinase activity of papain. The results showed that about 1 microg recombinant cystatin could inhibit the activity of 15 microg papain. Heat stability assay results showed that there was a decrease in inhibitory activity of cystatin with the increasing of temperature. When solution of recombinant cystatin was kept at 70 degrees C for 5min, the inhibitory activity reduced fast. While the recombinant cystatin was heated to 90 degrees C for 5min, the inhibitory activity of recombinant cystatin was undetected. The inhibitory activity for recombinant Chinese sturgeon cystatin was higher than that of CPI (cysteine proteinase inhibitor) from seeds of corn, that about 1 microg purified CIP could inhibited the activity of 0.278 microg papain. But the heat stability of recombinant cystatin is lower than that of the corn CPI. The expression level and the activity of recombinant cystatin from yeast Pichia pastoris were higher than those from E. coli. Moreover, recombinant cystatin from Pichia pastoris was easier to separate and purify. This paper reported that recombinant fish cystatin was produced in a highly efficient expression system based on the methylotrophic yeast, further work will focus on the function of recombinant Chinese sturgeon cystatin to resist fish disease and explore the value of cystatin as a food additive to inhibit cysteine proteinases during surimi processing.