Modularly assembled multiplex prime editors for simultaneous editing of agronomically important genes in rice.

Prime editing (PE) technology enables precise alterations in the genetic code of a genome of interest. PE offers great potential for identifying major agronomically important genes in plants and editing them into superior variants, ideally targeting multiple loci simultaneously to realize the collective effects of the edits. Here, we report the development of a modular assembly-based multiplex PE system in rice and demonstrate its efficacy in editing up to four genes in a single transformation experiment. The duplex PE (DPE) system achieved a co-editing efficiency of 46.1% in the T0 generation, converting TFIIAγ5 to xa5 and xa23 to Xa23SW11. The resulting double-mutant lines exhibited robust broad-spectrum resistance against multiple Xanthomonas oryzae pathovar oryzae (Xoo) strains in the T1 generation. In addition, we successfully edited OsEPSPS1 to an herbicide-tolerant variant and OsSWEET11a to a Xoo-resistant allele, achieving a co-editing rate of 57.14%. Furthermore, with the quadruple PE (QPE) system, we edited four genes-two for herbicide tolerance (OsEPSPS1 and OsALS1) and two for Xoo resistance (TFIIAγ5 and OsSWEET11a)-using one construct, with a co-editing efficiency of 43.5% for all four genes in the T0 generation. We performed multiplex PE using five more constructs, including two for triplex PE (TPE) and three for QPE, each targeting a different set of genes. The editing rates were dependent on the activity of pegRNA and/or ngRNA. For instance, optimization of ngRNA increased the PE rates for one of the targets (OsSPL13) from 0% to 30% but did not improve editing at another target (OsGS2). Overall, our modular assembly-based system yielded high PE rates and streamlined the cloning of PE reagents, making it feasible for more labs to utilize PE for their editing experiments. These findings have significant implications for advancing gene editing techniques in plants and may pave the way for future agricultural applications.

High-efficiency prime editing enables new strategies for broad-spectrum resistance to bacterial blight of rice.

Using genetic resistance against bacterial blight (BB) caused by Xanthomonas oryzae pathovar oryzae (Xoo) is a major objective in rice breeding programmes. Prime editing (PE) has the potential to create novel germplasm against Xoo. Here, we use an improved prime-editing system to implement two new strategies for BB resistance. Knock-in of TAL effector binding elements (EBE) derived from the BB susceptible gene SWEET14 into the promoter of a dysfunctional executor R gene xa23 reaches 47.2% with desired edits including biallelic editing at 18% in T0 generation that enables an inducible TALE-dependent BB resistance. Editing the transcription factor TFIIA gene TFIIAγ5 required for TAL effector-dependent BB susceptibility recapitulates the resistance of xa5 at an editing efficiency of 88.5% with biallelic editing rate of 30% in T0 generation. The engineered loci provided resistance against multiple Xoo strains in T1 generation. Whole-genome sequencing detected no OsMLH1dn-associated random mutations and no off-target editing demonstrating high specificity of this PE system. This is the first-ever report to use PE system to engineer resistance against biotic stress and to demonstrate knock-in of 30-nucleotides cis-regulatory element at high efficiency. The new strategies hold promises to fend rice off the evolving Xoo strains and protect it from epidemics.

Optimized prime editing efficiently generates heritable mutations in maize.

Low efficiency is the main obstacle to using prime editing in maize (Zea mays). Recently, prime-editing efficiency was greatly improved in mammalian cells and rice (Oryza sativa) plants by engineering prime-editing guide RNAs (pegRNAs), optimizing the prime editor (PE) protein, and manipulating cellular determinants of prime editing. In this study, we tested PEs optimized via these three strategies in maize. We demonstrated that the ePE5max system, composed of PEmax, epegRNAs (pegRNA-evopreQ. 1), nicking single guide RNAs (sgRNAs), and MLH1dn, efficiently generated heritable mutations that conferred resistance to herbicides that inhibit 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), acetolactate synthase (ALS), or acetyl CoA carboxylase (ACCase) activity. Collectively, we demonstrate that the ePE5max system has sufficient efficiency to generate heritable (homozygous or heterozygous) mutations in maize target genes and that the main obstacle to using PEs in maize has thus been removed.

Comprehensive quality evaluation of Polygoni Orientalis Fructus and its processed product: chemical fingerprinting and simultaneous determination of seven major components coupled with chemometric analyses.

INTRODUCTION: Polygoni Orientalis Fructus (POF) is a clinically effective Chinese medicine. Raw POF (RPOF) and POF Tostus (POFT) are used separately in clinics. However, incomplete progress has been made on quality control. OBJECTIVE: To establish a comprehensive method for quality assessment of RPOF and POFT and to discriminate these two varieties. METHODOLOGY: High-performance liquid chromatography combined with the diode array detector (HPLC-DAD) methods were developed for fingerprinting and quantitative analysis of seven major compounds in RPOF and POFT, and the main components were determined by HPLC-DAD coupled with Fourier-transform ion cyclotron resonance-mass spectrometry. Chemometric approaches were performed to discriminate RPOF and POFT and to screen discriminatory components. RESULTS: Fingerprints were established and 12 common peaks were identified, cannabisin G and cannabisin E were firstly identified from POF. In quantitative analysis, all analytes showed good regression (R > 0.9996) within test ranges and the recovery of the method was in the range 96.6-104.3%. Fingerprints in conjunction with similarity analysis and hierarchical clustering analysis (HCA) demonstrated the consistent quality of RPOF and showed a clear discrimination between RPOF and POFT. Principal component analysis, partial least-squares discriminant analysis, and heatmap-HCA on quantitative data not only gave a clear differentiation between RPOF and POFT, but they also suggested that quercetin, 3,5,7-trihydroxychromone, and N-trans-feruloyltyramine acted as the main factors responsible for the sample differences. CONCLUSIONS: Chromatographic analysis in combination with chemometric analysis provides a simple and reliable method of comparing and evaluating the qualities of RPOF and POFT.

Efficacy of uremic pruritus treatment in patients undergoing hemodialysis, a network meta-analysis for randomized clinical trials.

INTRODUCTION: Uremic pruritus is very common in hemodialysis or renal failure patients, there were lots of available treatments such as gabapentin, pregabalin, ondansetron, etc. However, there is no quantified study comparing these treatments together, it is impossible to conduct a clinical trial involving so many treatments, so we conduct a network meta-analysis to compare them. METHOD: We collected mean difference and standard error of visual analogue scale data as outcome. In total we collected 15 articles, 15 articles, 1180 subjects and 6 treatments included to this study. RESULTS: In these comparisons, gabapentin showed the largest effect MD: 5.19, 95%CI [3.77, 6.61], anti-histamine MD: 4.65, 95%CI [2.22, 7.07] and pregabalin MD: 4.62, 95%CI [2.71, 6.62] showed a similar effect. Opioid pathway related treatment also showed a significant but not so large effect MD: 2.45, 95%CI [0.41, 4.49]. Ondansetron and Doxepin didn't show a significant improvement among placebo, the overall quantifying heterogeneity I2=43.1%. There is no statically difference between gabapentin, pregabalin and anti-histamine treatments. CONCLUSIONS: So we conclude that gabapentin, pregabalin and anti-histamine has a similar efficacy on pruritus control.

Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize.

Prime editing is a novel and universal CRISPR/Cas-derived precision genome-editing technology that has been recently developed. However, low efficiency of prime editing has been shown in transgenic rice lines. We hypothesize that enhancing pegRNA expression could improve prime-editing efficiency. In this report, we describe two strategies for enhancing pegRNA expression. We construct a prime editing vector harboring two pegRNA variants for W542L and S621I double mutations in ZmALS1 and ZmALS2. Compared with previous reports in rice, we achieve much higher prime-editing efficiency in maize. Our results are inspiring and provide a direction for the optimization of plant prime editors.

The clinical effect and safety of the treatment of tibia intercondylar eminence fracture with cannulated screw and suture fixation under arthroscope: Protocol for a systematic review and meta-analysis of randomized controlled trials.

BACKGROUND: The clinical effects and safety over the treatment of tibia intercondylar eminence fracture (TIEF) with cannulated screw and suture fixation were evaluated under arthroscope systematically, providing evidence-based medical support for the selection of surgical methods in terms of minimally invasive arthroscopic treatment for TIEF. METHODS: The English databases of PubMed, EMBASE, Cochrane Library, CNKI, SinoMed, VIP, and Wanfang databases were searched by computer. The randomized controlled trials were conducted to compare the clinical effects of TIEF with cannulated screw and suture fixation under arthroscope. The retrieval period is from the beginning of database building to January 2020. There is no language restriction. Chinese databases are searched by keywords, while English databases are searched by the combination of subject words and free words. According to the retrieval strategy, the two evaluators will lead the conforming documents into Note Express for repeated literature screening, and the two evaluators will extract and cross-check the conforming documents according to the pre-designed data extraction table. Two researchers adopted the modified Jadad scale independently to evaluate the quality of the literature. The RevMan 5.3 version software provided by the Cochrane Collaboration Network was adopted for statistical analysis. RESULTS: The study will strictly review and extract the data included in the literature, and scientifically make statistical analysis for the pre-set outcome indicators. All the research processes will be conducted in strict accordance with the guidance of system evaluation. In this study, the differences between cannulated screw fixation and suture fixation under arthroscopy will be evaluated by comparing the relevant outcome indicators. All the results of this study will be published openly in a highly influential professional academic journal. CONCLUSION: The paper adopted Cochrane system evaluation method to collect and sort out the published literature about the treatment of tibial eminence fracture between cannulated screw fixation and suture fixation under arthroscopy, and to compare the clinical efficacy and safety of the two fixation methods utilizing meta-analysis and comparison of related outcome indicators. Through this study, we will draw a positive conclusion, which will provide a basis for the better treatment of tibial eminence fracture. PROSPERO REGISTRATION NUMBER: PROSPERO CRD42020168433.

Agrobacterium-mediated delivery of CRISPR/Cas reagents for genome editing in plants enters an era of ternary vector systems.

Lack of appropriate methods for delivery of genome-editing reagents is a major barrier to CRISPR/Cas-mediated genome editing in plants. Agrobacterium-mediated genetic transformation (AMGT) is the preferred method of CRISPR/Cas reagent delivery, and researchers have recently made great improvements to this process. In this article, we review the development of AMGT and AMGT-based delivery of CRISPR/Cas reagents. We give an overview of the development of AMGT vectors including binary vector, superbinary vector, dual binary vector, and ternary vector systems. We also review the progress in Agrobacterium genomics and Agrobacterium genetic engineering for optimal strains. We focus in particular on the ternary vector system and the resources we developed. In summary, it is our opinion that Agrobacterium-mediated CRISPR/Cas genome editing in plants is entering an era of ternary vector systems, which are often integrated with morphogenic regulators. The new vectors described in this article are available from Addgene and/or MolecularCloud for sharing with academic investigators for noncommercial research.

A Novel Ternary Vector System United with Morphogenic Genes Enhances CRISPR/Cas Delivery in Maize.

The lack of efficient delivery methods is a major barrier to clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-mediated genome editing in many plant species. Combinations of morphogenic regulator (MR) genes and ternary vector systems are promising solutions to this problem. In this study, we first demonstrated that MR vectors greatly enhance maize (Zea mays) transformation. We then tested a CRISPR/Cas9 MR vector in maize and found that the MR and CRISPR/Cas9 modules have no negative influence on each other. Finally, we developed a novel ternary vector system to integrate the MR and CRISPR/Cas modules. Our ternary vector system is composed of new pGreen-like binary vectors, here named pGreen3, and a pVS1-based virulence helper plasmid, which also functions as a replication helper for the pGreen3 vectors in Agrobacterium tumefaciens The pGreen3 vectors were derived from the plasmid pRK2 and display advantages over pGreen2 vectors regarding both compatibility and stability. We demonstrated that the union of our ternary vector system with MR gene modules has additive effects in enhancing maize transformation and that this enhancement is especially evident in the transformation of recalcitrant maize inbred lines. Collectively, our ternary vector system-based tools provide a user-friendly solution to the low efficiency of CRISPR/Cas delivery in maize and represent a basic platform for developing efficient delivery tools to use in other plant species recalcitrant to transformation.

Potential high-frequency off-target mutagenesis induced by CRISPR/Cas9 in Arabidopsis and its prevention.

KEY MESSAGE: We present novel observations of high-specificity SpCas9 variants, sgRNA expression strategies based on mutant sgRNA scaffold and tRNA processing system, and CRISPR/Cas9-mediated T-DNA integrations. Specificity of CRISPR/Cas9 tools has been a major concern along with the reports of their successful applications. We report unexpected observations of high frequency off-target mutagenesis induced by CRISPR/Cas9 in T1 Arabidopsis mutants although the sgRNA was predicted to have a high specificity score. We also present evidence that the off-target effects were further exacerbated in the T2 progeny. To prevent the off-target effects, we tested and optimized two strategies in Arabidopsis, including introduction of a mCherry cassette for a simple and reliable isolation of Cas9-free mutants and the use of highly specific mutant SpCas9 variants. Optimization of the mCherry vectors and subsequent validation found that fusion of tRNA with the mutant rather than the original sgRNA scaffold significantly improves editing efficiency. We then examined the editing efficiency of eight high-specificity SpCas9 variants in combination with the improved tRNA-sgRNA fusion strategy. Our results suggest that highly specific SpCas9 variants require a higher level of expression than their wild-type counterpart to maintain high editing efficiency. Additionally, we demonstrate that T-DNA can be inserted into the cleavage sites of CRISPR/Cas9 targets with high frequency. Altogether, our results suggest that in plants, continuous attention should be paid to off-target effects induced by CRISPR/Cas9 in current and subsequent generations, and that the tools optimized in this report will be useful in improving genome editing efficiency and specificity in plants and other organisms.

MISSA 2.0: an updated synthetic biology toolbox for assembly of orthogonal CRISPR/Cas systems.

Efficient generation of plants carrying mutations in multiple genes remains a challenge. Using two or more orthogonal CRISPR/Cas systems can generate plants with multi-gene mutations, but assembly of these systems requires a robust, high-capacity toolkit. Here, we describe MISSA 2.0 (multiple-round in vivo site-specific assembly 2.0), an extensively updated toolkit for assembly of two or more CRISPR/Cas systems. We developed a novel suicide donor vector system based on plasmid RK2, which has much higher cloning capacity than the original, plasmid R6K-based system. We validated the utility of MISSA 2.0 by assembling multiple DNA fragments into the E. coli chromosome, and by creating transgenic Arabidopsis thaliana that constitutively or inducibly overexpress multiple genes. We then demonstrated that the higher cloning capacity of the RK2-derived MISSA 2.0 donor vectors facilitated the assembly of two orthogonal CRISPR/Cas systems including SpCas9 and SaCas9, and thus facilitated the creation of transgenic lines harboring these systems. We anticipate that MISSA 2.0 will enable substantial advancements in multiplex genome editing based on two or more orthogonal CRISPR/Cas9 systems, as well as in plant synthetic biology.

Hydroxyethyl starch resuscitation downregulate pro-inflammatory cytokines in the early phase of severe acute pancreatitis: A retrospective study.

In the present study, we investigated the effects of hydroxyethyl starch (HES) 130/0.4 on serum pro-inflammatory variables, immunologic variables, fluid balance (FB)-negative(-) rate and renal function in severe acute pancreatitis (SAP) patients. From October, 2007 to November, 2008, a total of 120 SAP patients were enrolled in this retrospective study. Fifty-nine patients in the HES group received 6% HES 130/0.4 combined with crystalloid solution for fluid resuscitation (HES group). In the control group, 61 patients received only crystalloid solution after admission. Interleukin (IL)-1, IL-6, IL-8 and tumor necrosis factor (TNF)-α levels in serum were measured on days 1, 2, 4 and 8. The peripheral blood CD4+CD8+ T lymphocyte rates, serum BUN and Cr values were also measured on days 1, 4 and 8. Patients with FB(-) rates were recorded from day 1 to 8. Interaction term analysis (hospital stay and fluid resuscitation methods) based on mixed-effects regression model revealed significantly lower levels of IL-1 and TNF-α in the HES group compared with the control group. The difference in curve's risk ratio was not significant for IL-6, CD4+CD8+ T lymphocyte rate, BUN and Cr values (P>0.05). In the HES group, we detected a significantly higher rate of patients with FB(-) from day 4 to 8 (P<0.05). Thus, HES 130/0.4 resuscitation could decrease the IL-1 and IL-8 levels, shorten the duration of positive FB, and preserve the patient's immune status as well as renal function during the early phase of SAP.

The Transcription Factor AtDOF4.7 Is Involved in Ethylene- and IDA-Mediated Organ Abscission in Arabidopsis.

Organ abscission is an important plant developmental process that occurs in response to environmental stress or pathogens. In Arabidopsis, ligand signals, such as ethylene or INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), can regulate organ abscission. Previously, we reported that overexpression of AtDOF4.7, a transcription factor gene, directly suppresses the expression of the abscission-related gene ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE 2 (ADPG2), resulting in a deficiency of floral organ abscission. However, the relationship between AtDOF4.7 and abscission pathways still needs to be investigated. In this study, we showed that ethylene regulates the expression of AtDOF4.7, and the peptide ligand, IDA negatively regulates AtDOF4.7 at the transcriptional level. Genetic evidence indicates that AtDOF4.7 and IDA are involved in a common pathway, and a MAPK cascade can phosphorylate AtDOF4.7 in vitro. Further in vivo data suggest that AtDOF4.7 protein levels may be regulated by this phosphorylation. Collectively, our results indicate that ethylene regulates AtDOF4.7 that is involved in the IDA-mediated floral organ abscission pathway.

Is there comparable morbidity in pylorus-preserving and pylorus-resecting pancreaticoduodenectomy? A meta-analysis.

Pancreaticoduodenectomy (PD) is the most effective treatment for patients with pancreatic head or periampullary lesions. Two major strategies exist: pylorus-preserving pancreaticoduodenectomy (PPPD) and pylorus-resecting pancreaticoduodenectomy (PRPD). However, it is yet unclear regarding the morbidity after PPPD and PRPD. This study analyzed the morbidity after PPPD and PRPD to determine the optimal surgical treatment of masses in the pancreatic head or periampullary region. A systematic search of databases identifying randomized controlled trials (RCTs) from the Cochrane Library, PubMed, EMBASE and Web of Science was performed. Outcome was compared by postoperative morbidity including overall morbidity, pancreatic fistulas, wound infections, postoperative bleeding, biliary leakage, ascites and delayed gastric emptying (DGE) rate between PPPD and PRPD. The DGE rate in the PRPD subgroups (conventional PD [CPD] and subtotal stomach-preserving PD [SSPPD], respectively) was also analyzed. The results showed that 9 RCTs including 722 participants were included for meta-analysis. Among these RCTs, 7 manuscripts described PRPD as CPD, and 2 manuscripts described PRPD as SSPPD. There were no significant differences in the overall morbidity, pancreatic fistulas, wound infections, postoperative bleeding, or biliary leakage between PPPD and PRPD. There was a lower rate of DGE with PRPD than that with PPPD (RR=2.15, P=0.03, 95% CI, 1.09-4.23). Further subgroup analysis indicated a comparable DGE rate for the CPD but a lower DGE rate for the SSPPD group than the PPPD group. However, the result did not indicate any difference between CPD and SSPPD regarding the DGE rate (P=0.92). It is suggested that PPPD is comparable to PRPD in overall morbidity, pancreatic fistulas, wound infections, postoperative bleeding and biliary leakage. The current data are not sufficient to draw a conclusion regarding which surgical procedure is associated with a lower postoperative DGE rate. Our conclusions were limited by the available data. Further evaluations of RCTs are needed.

Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation.

Arabidopsis mutants produced by constitutive overexpression of the CRISPR/Cas9 genome editing system are usually mosaics in the T1 generation. In this study, we used egg cell-specific promoters to drive the expression of Cas9 and obtained non-mosaic T1 mutants for multiple target genes with high efficiency. Comparisons of 12 combinations of eight promoters and two terminators found that the efficiency of the egg cell-specific promoter-controlled CRISPR/Cas9 system depended on the presence of a suitable terminator, and the composite promoter generated by fusing two egg cell-specific promoters resulted in much higher efficiency of mutation in the T1 generation compared with the single promoters.

A CRISPR/Cas9 toolkit for multiplex genome editing in plants.

BACKGROUND: To accelerate the application of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) system to a variety of plant species, a toolkit with additional plant selectable markers, more gRNA modules, and easier methods for the assembly of one or more gRNA expression cassettes is required. RESULTS: We developed a CRISPR/Cas9 binary vector set based on the pGreen or pCAMBIA backbone, as well as a gRNA (guide RNA) module vector set, as a toolkit for multiplex genome editing in plants. This toolkit requires no restriction enzymes besides BsaI to generate final constructs harboring maize-codon optimized Cas9 and one or more gRNAs with high efficiency in as little as one cloning step. The toolkit was validated using maize protoplasts, transgenic maize lines, and transgenic Arabidopsis lines and was shown to exhibit high efficiency and specificity. More importantly, using this toolkit, targeted mutations of three Arabidopsis genes were detected in transgenic seedlings of the T1 generation. Moreover, the multiple-gene mutations could be inherited by the next generation. CONCLUSIONS: We developed a toolkit that facilitates transient or stable expression of the CRISPR/Cas9 system in a variety of plant species, which will facilitate plant research, as it enables high efficiency generation of mutants bearing multiple gene mutations.

Pylorus-preserving versus pylorus-resecting pancreaticoduodenectomy for periampullary and pancreatic carcinoma: a meta-analysis.

BACKGROUND: The aim of this meta-analysis was to compare the long-term survival, mortality, morbidity and the operation-related events in patients with periampullary and pancreatic carcinoma undergoing pylorus-preserving pancreaticoduodenectomy (PPPD) and pylorus-resecting pancreaticoduodenectomy (PRPD). METHOD: A systematic search of literature databases (Cochrane Library, PubMed, EMBASE and Web of Science) was performed to identify studies. Outcome measures comparing PPPD versus PRPD for periampullary and pancreatic carcinoma were long-term survival, mortality, morbidity (overall morbidity, delayed gastric emptying [DGE], pancreatic fistula, wound infection, postoperative bleeding, biliary leakage, ascites and gastroenterostomy leakage) and operation related events (hospital stays, operating time, intraoperative blood loss and red blood cell transfusions). RESULTS: Eight randomized controlled trials (RCTs) including 622 patients were identified and included in the analysis. Among these patients, it revealed no difference in long-term survival between the PPPD and PRPD groups (HR = 0.23, p = 0.11). There was a lower rate of DGE (RR = 2.35, p = 0.04, 95% CI, 1.06-5.21) with PRPD. Mortality, overall morbidity, pancreatic fistula, wound infection, postoperative bleeding, biliary leakage, ascites and gastroenterostomy leakage were not significantly different between the groups. PPPDs were performed more quickly than PRPDs (WMD = 53.25 minutes, p = 0.01, 95% CI, 12.53-93.97); and there was less estimated intraoperative blood loss (WMD = 365.21 ml, p = 0.006, 95% CI, 102.71-627.71) and fewer red blood cell transfusions (WMD = 0.29 U, p = 0.003, 95% CI, 0.10-0.48) in patients undergoing PPPD. The hospital stays showed no significant difference. CONCLUSIONS: PPPD had advantages over PRPD in operating time, intraoperative blood loss and red blood cell transfusions, but had a significantly higher rate of DGE for periampullary and pancreatic carcinoma. PPPD and PRPD had comparable mortality and morbidity including pancreatic fistulas, wound infections, postoperative bleeding, biliary leakage, ascites and gastroenterostomy leakage. Our conclusions were limited by the available data. Further evaluations of high-quality RCTs are needed.

[The binding property of different regions in Duffy-binding-like domain alpha of Plasmodium falciparum erythrocyte membrane protein 1 to heparin].

OBJECTIVE: To clone and express Plasmodium falciparum erythrocyte membrane protein 1 DBLalpha (PfEMP1-DBLalpha) and three fragments genes, and screen the strongest affinity sequence with the red blood cell surface receptors-heparin or heparin sulfate in the structure of PfEMP1-DBLalpha. METHODS: The sequence of PfEMP1-DBLalpha1245 was optimized according to the characteristics of E. coli codon, synthesized, and divided into three fragments (DBLaA, DBLalphaB, and DBLalphaC) by PCR. Full-length gene and three gene fragments were subcloned into PGEX-4T-1 vector, and transformed into E. coli BL21 and then induced with IPTG for expression. The recombinant protein was purified from bacterial lysates using glutathione-Sepharose 4B. Heparin affinity test and glycosaminoglycan (GAG) inhibition test were used to analyze the affinity between recombinant protein and heparin. RESULTS: Four recombinant proteins(DBLalpha1245, DBLalphaA, DBLalphaB, and DBLalphaC) were expressed as solubility and the relative molecular weight (M(r) 73 600, M(r) 41 600, M(r) 42 500, and M(r) 41 500) were conformed to the prediction size. Heparin affinity test and GAG inhibition test showed that the four recombinant proteins were binded to the heparin-Sepharose, but not for the GST control. DBLalphaC (Q285-Y415) had the strongest affinity to heparin. CONCLUSION: The strongest affinity sequence with heparin or heparin sulfate in the structure of PfEMP1-DBLalpha is Q285-Y415, which plays a role in binding of Plasmodium falciparum infected red blood cells to the peripheral red blood cells.