Efficient assembly and release of SARS coronavirus-like particles by a heterologous expression system

Virus-like particles (VLPs) produced by recombinant expression of the major viral structural proteins could be an attractive method for severe acute respiratory syndrome (SARS) control. In this study, using the baculovirus system, we generated recombinant viruses that expressed S, E, M and N structural proteins of SARS-CoV either individually or simultaneously. The expression level, size and authenticity of each recombinant SARS-CoV protein were determined. In addition, immunofluorescence and FACS analysis confirmed the cell surface expression of the S protein. Co-infections of insect cells with two recombinant viruses demonstrated that M and E could assemble readily to form smooth surfaced VLPs. On the other hand, simultaneous high level expression of S, E and M by a single recombinant virus allowed the very efficient assembly and release of VLPs. These data demonstrate that the VLPs are morphological mimics of virion particles. The high level expression of VLPs with correct S protein conformation by a single recombinant baculovirus offers a potential candidate vaccine for SARS.     

Cloning and optimization of induction conditions for mature PsaA (pneumococcal surface adhesin A) expression in Escherichia coli and recombinant protein stability during long-term storage

The gene corresponding to mature PsaA from Streptococcus pneumoniae serotype 14 was cloned into a plasmid with kanamycin resistance and without a purification tag in Escherichia coli to express high levels of the recombinant protein for large-scale production as a potential vaccine candidate or as a carrier for polysaccharide conjugation at Bio-Manguinhos/Fiocruz. The evaluation of induction conditions (IPTG concentration, temperature and time) in E. coli was accomplished by experimental design techniques to enhance the expression level of mature recombinant PsaA (rPsaA). The optimization of induction process conditions led us to perform the recombinant protein induction at 25°C for 16 h, with 0.1mM IPTG in Terrific Broth medium. At these conditions, the level of mature rPsaA expression obtained in E. coli BL21 (DE3) Star by pET28a induction with IPTG was in the range of 0.8 g/L of culture medium, with a 10-fold lower concentration of inducer than usually employed, which contributes to a less expensive process. Mature rPsaA expressed in E. coli BL21 (DE3) Star accounted for approximately 30-35% of the total protein. rPsaA purification by ion exchange allowed the production of high-purity recombinant protein without fusion tags. The results presented in this work confirm that the purified recombinant protein maintains its stability and integrity for long periods of time in various storage conditions (temperatures of 4 or -70°C using different cryoprotectors) and for at least 3 years at 4 or -70°C in PBS. The conformation of the stored protein was confirmed using circular dichroism. Mature rPsaA antigenicity was proven by anti-rPsaA mouse serum recognition through western blot analysis, and no protein degradation was detected after long periods of storage.     

Effects of the number of amino acid residues in the signal segment upstream or downstream of the NS2B-3 cleavage site on production and secretion of prM/M-E virus-like particles of West Nile virus

Expression of genes for precursor M (prM) and envelope (E) proteins of West Nile virus (WNV) leads to the production of small, capsidless, and non-infectious virus-like particles (VLPs) possessing the E antigen which is responsible for viral entry and immune protection. It has been reported that processing of the secretion signal affects viral release. We examined the secretion efficiency of VLPs into the culture medium from RK13 or 293 T cells transfected with expression vectors for prM and E proteins of WNV which were constructed to comprise different lengths of signal peptides upstream of the prM-E domain. The number of amino acid residues present in the segment markedly affected the production, processing, and secretion of VLPs. Secreted VLPs possessed both the processed M protein and the glycosylated E protein. In addition, immunization with VLPs induced neutralizing antibodies in C3H/HeN mice. These results indicate that the number of amino acid residues comprising the N-terminus of the signal segment controls the efficiency of assembly, maturation, and release of VLPs in the absence of viral protease, which in turn indicates the potential of VLPs as a candidate for an effective WNV subunit vaccine.     

Cultivation at 6-10°C is an effective strategy to overcome the insolubility of recombinant proteins in Escherichia coli

Protein expression in Escherichia coli at 15-25°C is widely used to increase the solubility of recombinant proteins. However, many recombinant proteins are insolubly expressed even at those low temperatures. Here, we show that recombinant proteins can be expressed as soluble forms by simply lowering temperature to 6-10°C without cold adapted chaperon systems. By using E. coli Rosetta-gami2(DE3), we obtained 1.8 and 0.9mg of Cryptopygus antarticus mannanase (CaMan) and cellulase (CaCel) from 1l culture grown at 6 and 10°C, respectively. Cultivation at 10°C also led to successful expression of EM3L7 (a lipase isolated from a metagenomic library) in a soluble form in E. coli BL21(DE3). Consequently, E. coli cultivation at 6-10°C is an effective strategy for overcoming a major hurdle of the inclusion body formation.     

大肠杆菌表达的病毒样颗粒疫苗

重组病毒样颗粒是病毒衣壳蛋白外源表达的重要形式,形态结构与天然病毒高度相似,位于纳米尺度的大小易于被免疫系统识别,可激发机体产生保护性免疫反应,且不含有病毒基因,因此,是一种理想的疫苗形式,也是基于结构进行疫苗设计的重要结构载体。目前已上市的乙型肝炎疫苗、人乳头瘤病毒疫苗和戊型肝炎疫苗等基因工程疫苗均采用病毒样颗粒形式。大肠杆菌表达系统被广泛用于基因工程药物的生产,具有安全性好、生产周期短、易于放大生产等优点,在病毒样颗粒疫苗应用上具有良好前景。本文综述了利用大肠杆菌研制戊型肝炎疫苗和人乳头瘤病毒疫苗的进展,特别是这些病毒样颗粒疫苗的表达及组装、表位结构特征和临床试验结果。     

Analysis of antigenicity and topology of E2 glycoprotein present on recombinant hepatitis C virus-like particles

Purification of hepatitis C virus (HCV) from sera of infected patients has proven elusive, hampering efforts to perform structure-function analysis of the viral components. Recombinant forms of the viral glycoproteins have been used instead for functional studies, but uncertainty exists as to whether they closely mimic the virion proteins. Here, we used HCV virus-like particles (VLPs) generated in insect cells infected with a recombinant baculovirus expressing viral structural proteins. Electron microscopic analysis revealed a population of pleomorphic VLPs that were at least partially enveloped with bilayer membranes and had viral glycoprotein spikes protruding from the surface. Immunogold labeling using specific monoclonal antibodies (MAbs) demonstrated these protrusions to be the E1 and E2 glycoproteins. A panel of anti-E2 MAbs was used to probe the surface topology of E2 on the VLPs and to compare the antigenicity of the VLPs with that of truncated E2 (E2(660)) or the full-length (FL) E1E2 complex expressed in mammalian cells. While most MAbs bound to all forms of antigen, a number of others showed striking differences in their abilities to recognize the various E2 forms. All MAbs directed against hypervariable region 1 (HVR-1) recognized both native and denatured E2(660) with comparable affinities, but most bound either weakly or not at all to the FL E1E2 complex or to VLPs. HVR-1 on VLPs was accessible to these MAbs only after denaturation. Importantly, a subset of MAbs specific for amino acids 464 to 475 and 524 to 535 recognized E2(660) but not VLPs or FL E1E2 complex. The antigenic differences between E2(660,) FL E1E2, and VLPs strongly point to the existence of structural differences, which may have functional relevance. Trypsin treatment of VLPs removed the N-terminal part of E2, resulting in a 42-kDa fragment. In the presence of detergent, this was further reduced to a trypsin-resistant 25-kDa fragment, which could be useful for structural studies.     

A mathematical model of mitochondrial swelling

Background

Salmonid alphavirus (SAV) is a widespread pathogen in European aquaculture of salmonid fish. Distinct viral subtypes have been suggested based on sequence comparisons and some of these have different geographical distributions. In Norway, only SAV subtype 3 have so far been identified. Little is known about viral mechanisms important for pathogenesis and transmission. Tools for detailed exploration of SAV genomes are therefore needed.

Results

Infectious cDNA clones in which a genome of subtype 3 SAV is under the control of a CMV promoter were constructed. The clones were designed to express proteins that are putatively identical to those previously reported for the SAVH20/03 strain. A polyclonal antiserum was raised against a part of the E2 glycoprotein in order to detect expression of the subgenomic open reading frame (ORF) encoding structural viral proteins. Transfection of the cDNA clone revealed the expression of the E2 protein by IFAT, and in serial passages of the supernatant the presence of infectious recombinant virus was confirmed through RT-PCR, IFAT and the development of a cytopathic effect similar to that seen during infection with wild type SAV. Confirmation that the recovered virus originated from the infectious plasmid was done by sequence identification of an introduced genetic tag. The recombinant virus was infectious also when an additional ORF encoding an EGFP reporter gene under the control of a second subgenomic alphavirus promoter was added. Finally, we used the system to study the effect of selected point mutations on infectivity in Chinook salmon embryo cells. While introduced mutations in nsP2₁₉₇, nsP3₂₆₃ and nsP3₃₂₃ severely reduced infectivity, a serine to proline mutation in E2₂₀₆ appeared to enhance the virus titer production.

Conclusion

We have constructed infectious clones for SAV based on a subtype 3 genome. The clones may serve as a platform for further functional studies.     

牛病毒性腹泻病毒1型病毒样颗粒的制备及其对豚鼠的免疫原性分析

为了获得预防牛病毒性腹泻病毒1型(bovine viral diarrhea virus 1,BVDV-1)感染的病毒样颗粒,扩增C-Ems-E1-E2编码区段并克隆至pFastBacDaul载体,转化大肠杆菌DH10Bac感受态细胞与Bacmid重组获得Bacmid-BVDV-1,转染至Sf9细胞,获得重组杆状病毒B...     

Effect of the DnaK chaperone on the conformational quality of JCV VP1 virus‐like particles produced in Escherichia coli

Protein nanoparticles such as virus‐like particles (VLPs) can be obtained by recombinant protein production of viral capsid proteins and spontaneous self‐assembling in cell factories. Contrarily to infective viral particles, VLPs lack infective viral genome while retaining important viral properties like cellular tropism and intracellular delivery of internalized molecules. These properties make VLPs promising and fully biocompatible nanovehicles for drug delivery. VLPs of human JC virus (hJCV) VP1 capsid protein produced in Escherichia coli elicit variable hemagglutination properties when incubated at different NaCl concentrations and pH conditions, being optimal at 200 mM NaCl and at pH range between 5.8 and 7.5. In addition, the presence or absence of chaperone DnaK in E. coli cells influence the solubility of recombinant VP1 and the conformational quality of this protein in the VLPs. The hemagglutination ability of hJCV VP1 VLPs contained in E. coli cell extracts can be modulated by buffer composition in the hemagglutination assay. It has been also determined that the production of recombinant hJCV VP1 in E. coli is favored by the absence of chaperone DnaK as observed by Western Blot analysis in different E. coli genetic backgrounds, indicating a proteolysis targeting role for DnaK. However, solubility is highly compromised in a DnaK^? E. coli strain suggesting an important role of this chaperone in reduction of protein aggregates. Finally, hemagglutination efficiency of recombinant VP1 is directly related to the presence of DnaK in the producing cells. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:744–748, 2014     

A 6K-Deletion Variant of Salmonid Alphavirus Is Non-Viable but Can Be Rescued through RNA Recombination

Pancreas disease (PD) of Atlantic salmon is an emerging disease caused by Salmonid alphavirus (SAV) which mainly affects salmonid aquaculture in Western Europe. Although genome structure of SAV has been characterized and each individual viral protein has been identified, the role of 6K protein in viral replication and infectivity remains undefined. The 6K protein of alphaviruses is a small and hydrophobic protein which is involved in membrane permeabilization, protein processing and virus budding. Because these common features are shared across many viral species, they have been named viroporins. In the present study, we applied reverse genetics to generate SAV3 6K-deleted (Δ6K) variant and investigate the role of 6K protein. Our findings show that the 6K-deletion variant of salmonid alphavirus is non-viable. Despite viral proteins of Δ6K variant are detected in the cytoplasm by immunostaining, they are not found on the cell surface. Further, analysis of viral proteins produced in Δ6K cDNA clone transfected cells using radioimmunoprecipitation (RIPA) and western blot showed a protein band of larger size than E2 of wild-type SAV3. When Δ6K cDNA was co-transfected with SAV3 helper cDNA encoding the whole structural genes including 6K, the infectivity was rescued. The development of CPE after co-transfection and resolved genome sequence of rescued virus confirmed full-length viral genome being generated through RNA recombination. The discovery of the important role of the 6K protein in virus production provides a new possibility for the development of antiviral intervention which is highly needed to control SAV infection in salmonids.     

Different applications of virus‐like particles in biology and medicine: Vaccination and delivery systems

Virus‐like particles (VLPs) mimic the whole construct of virus particles devoid of viral genome as used in subunit vaccine design. VLPs can elicit efficient protective immunity as direct immunogens compared to soluble antigens co‐administered with adjuvants in several booster injections. Up to now, several prokaryotic and eukaryotic systems such as insect, yeast, plant, and E. coli were used to express recombinant proteins, especially for VLP production. Recent studies are also generating VLPs in plants using different transient expression vectors for edible vaccines. VLPs and viral particles have been applied for different functions such as gene therapy, vaccination, nanotechnology, and diagnostics. Herein, we describe VLP production in different systems as well as its applications in biology and medicine. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 113–132, 2016.     

Life table studies of Elasmopalpus lignosellus (Lepidoptera: Pyralidae) on sugarcane

The lesser cornstalk borer, Elasmopalpus lignosellus (Zeller) (Lepidoptera: Pyralidae) is an important pest of sugarcane (a complex hybrid of Saccharum spp.) in southern Florida. Reproductive and life table parameters for E. lignosellus were examined at nine constant temperatures from 13 to 36 °C with sugarcane as the larval food source. The pre- and postoviposition periods decreased with increasing temperatures and reached their minimums at 33 and 36 °C, respectively. The oviposition period was longest at 27 °C. The mean fecundity, stage-specific survival, stage-specific fecundity, intrinsic rate of increase, and finite rate of increase were greatest at 30 °C and decreased with increasing or decreasing temperature. The net reproductive rate was greatest at 27 °C. The Logan-6 model best described the relationship between temperature and intrinsic rate of increase. The generation and population doubling times were longest at 13 and shortest at 33 and 30 °C, respectively. The most favorable temperatures for E. lignosellus population growth were between 27 and 33°C. Life table parameters for E. lignosellus reared on sugarcane were greater than for the Mexican rice borer [Eoreuma loftini (Dyar) (Lepidoptera: Crambidae)] reared on an artificial diet at 30 °C. The intrinsic rates of increase for the sugarcane borer [Diatraea saccharalis (F.) (Lepidoptera: Crambidae)] reared on sugarcane or corn were the same as for E. lignosellus reared on sugarcane at 27 °C, but the net reproductive rate was four times higher for the former than the latter borer species.     

Mapping of positive selection sites in the HIV-1 genome in the context of RNA and protein structural constraints

Background

Viral genomes of the human endogenous retrovirus K (HERV-K) family are integrated into the human chromosome and are transmitted vertically as Mendelian genes. Although viral particles are released by some transformed cells, they have never been shown to be infectious. In general, gammaretroviruses are produced as immature viral particles by accumulation of the Gag polyproteins at the plasma membrane, which subsequently bud from the cell surface. After release from the cell, Gag is further processed by proteolytic cleavage by the viral protease (PR), which results in morphologically mature particles with condensed cores. The HERV-K Gag polyprotein processing and function has not yet been precisely determined.

Results

We generated a recombinant poxvirus, encoding the human endogenous retrovirus K consensus gag-pro-pol genes (MVA-HERV-K_con) and obtained high levels of HERV-K Gag expression. The resulting retroviral particle assembled at the plasma membrane, as is typical for gammaretroviruses; and immature as well as mature retrovirus-like particles (VLPs) were observed around the infected cells. VLPs were purified, concentrated and separated by two-dimensional gel electrophoresis. The HERV-K Gag fragments were identified by mass spectroscopy and N-terminal sequencing which revealed that HERV-K Gag is processed into MA, a short spacer peptide, p15, CA and NC.

Conclusion

The cleavage sites of HERV-K Gag were mapped and found to be highly conserved among HERV-K genomes. The consensus HERV-K gag gene used in this study is known to support viral, infectivity [1], and thus the cleavage sites that were mapped in this study for all the Gag components are relevant for HERV-K infectivity.     

戊型肝炎病毒样颗粒在重组汉逊酵母中的发酵表达、纯化及其免疫原性分析

为了研制戊型肝炎新型基因工程疫苗,利用汉逊酵母表达系统表达重组戊型肝炎病毒样颗粒,成功构建了重组戊型肝炎疫苗工程菌株HP/HEV2.3,对该菌株的发酵条件和纯化工艺进行了研究。先将工作种子批进行发酵培养,收集发酵后的细胞培养物;对其先后进行细胞破碎、澄清和超滤、硅胶吸附和解吸附、超滤浓缩换液、色谱纯化及除菌过滤,制得重组汉逊酵母戊型肝炎病毒样颗粒,纯化收率为33%,纯度达99%;电镜观察显示该重组汉逊酵母戊型肝炎病毒样颗粒与天然戊型肝炎病毒颗粒理论大小一致,为32 nm;基因序列与理论一致;SDS-PAGE分析结果表明其表达的外源蛋白质分子量与预期的目的蛋白质分子量大小一致,均为56 k Da,表达量占细胞总蛋白的26%,表达水平为1.0 g/L发酵液;Western blotting、ELISA活性检测及小鼠免疫接种效力试验ED_(50)结果表明,此重组汉逊酵母戊型肝炎病毒样颗粒具有良好的抗原性和免疫原性,可用于制造戊型肝炎新型基因工程疫苗。     

Non-histone proteins and long-range organization of HeLa interphase DNA

We have studied the association of the Sindbis virus glycoproteins in mature virions and infected cells. The glycoproteins were cross-linked with bifunctional amino-reactive reagents (11 Å cross-linking distance), some of which could be subsequently cleaved by reduction. Using monospecific rabbit antisera against each viral envelope glycoprotein it was found that >90% of the cross-linked glycoprotein dimers from intact virions or virions solubilized with Triton X100 prior to cross-linking were heterodimers of E1 and E2. The pattern of cross-linked glycoproteins from intact virions as well as infected cells suggested that three E1-E2 dimers may be associated to form a hexameric subunit. Cross-linking of pulselabeled infected monolayers showed that PE2 was cross-linked to E1 less efficiently than was E2, suggesting that if PE2 and E1 are associated as a complex in infected cells then their conformation with respect to reactive amino groups is distinct from that of the mature virion glycoproteins. ts mutants of Sindbis virus in the complementation groups corresponding to E1 and PE2 fail to cleave PE2 at the non-permissive temperature (40 °C). In monolayers infected with these mutants or a heat-resistant variant of Sindbis virus, the glycoprotein precursors synthesized at the elevated temperature were readily cross-linked into large aggregates, indicating a temperature-sensitive tendency for the proteins to aggregate.     

Detection of salmonid alphavirus RNA in wild marine fish: implications for the origins of salmon pancreas disease in aquaculture

Salmonid alphaviruses (SAVs), which include the aetiological agents of salmon pancreas disease (SPD) in farmed Atlantic salmon Salmo salar L. and sleeping disease (SD) in rainbow trout Oncorhynchus mykiss (Walbaum), are significant viral pathogens of European salmonid aquaculture. SAV is horizontally transmitted and the virus can survive for extended periods in seawater. A lack of convincing evidence for vertical transmission coupled to the fact that the SPD virus (SPDV) occurs in historically infected sites irrespective of fallow period duration suggests that a substantial reservoir of infection exists in the marine environment. We used a highly sensitive real-time PCR (qPCR) assay targeting a region of the SAV nsP1 gene to screen wild marine fish species for the presence of SAV in an attempt to identify such a potential reservoir. Screened fish species were caught in the vicinity of aquaculture activity in an area with a previous history of SAV infection (Shetland Isles, Scotland). SAV RNA was detected in internal organs (kidney and heart) from the flatfish species common dab Limanda limanda, long rough dab Hippoglossoides platessoides, and plaice Pleuronectes platessa. Based on these findings, sampling was extended to an area remote from aquaculture activity (Stonehaven Bay, NE coast of Scotland) from where heart tissues obtained from common dab also tested positive. While no virus could be cultivated from these samples, qPCR detections were shown to be SAV-specific by sequencing of an alternative gene region (E2) to that targeted by the qPCR assay. Analysis of these nucleotide sequences revealed minor differences to those previously obtained from farmed salmon, and subsequent phylogenetic analysis of an E2 dataset demonstrated a subtype V-like sequence.     

溶氧反馈分批补料高密度培养人骨形成蛋白-2工程菌

对表达人骨形成蛋白－2成熟肽的基因工程大肠杆菌E.coli DH5α/pDH-B2m在500mL摇瓶中进行了培养条件的摸索实验,并在此基础上扩大至NBS Bioflo IV20L发酵罐,利用溶氧反馈－分批补料培养技术：在培养过程中保持适当的溶解氧（40％）,以溶氧值在线反馈控制搅拌速度及流加补料培养基,使细菌保持适当的比生长率,成功地进行了工程菌的高密度培养,最终菌体密度达OD600＝57,每升干菌量22.8g,目的蛋白的表达量占细菌总蛋白的30％,人骨形成蛋白－2成熟肽的理论产率达到3.59g/L。     

Molecular Cloning of Giant Panda Pituitary Prolactin cDNA and Its Expression in Escherichia coli

cDNA encoding pituitary (PRL) of giant panda was obtained using RT-PCR and expressed in E. coli. The results revealed that panda PRL cDNA encodes a precursor protein of 229 amino acids including a putative signal peptide of 30 amino acids and a mature protein of 199 residues with one potential N-glycosylation site. Sequence comparison indicated that panda PRL shares a high degree of identity to other known PRL sequences ranging from 98% with mink PRL to about 50% with rodent PRL. Six cysteine residues and 29 conserved residues distributed in four domains (PD1, PD2, PD3, and PD4) of PRL were observed through multiple sequence alignment. Fourteen key residues of binding sites 1 and 2 involved in receptor binding are conserved in panda PRL. GST fused recombinant panda PRL protein was efficiently expressed with the form of insoluble inclusion bodies in E. coli BL21 transformed with a pGEX-4T-1 expression vector containing the DNA sequence encoding mature panda PRL. Western blot analysis indicated that GST-panda PRL recombinant protein could be recognized by antibody against human PRL. Our results would contribute to further elucidating the structural and functional characteristics of pituitary PRL and provide a basis for the production of recombinant panda prolactin for future use in the breeding of giant panda.     

Efficient production of Japanese encephalitis virus-like particles by recombinant lepidopteran insect cells

The production of Japanese encephalitis (JE) virus-like particles (VLPs) in stably transformed lepidopteran insect cells was investigated. The DNA fragment encoding the JE virus (JEV) prM signal peptide, the precursor (prM) of the viral membrane protein (M), and the envelope glycoprotein (E) was cloned into the plasmid vector pIHAbla. The pIHAbla contained the Bombyx mori actin promoter downstream of the B. mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer for high-level expression, together with a blasticidin resistance gene for use as a selectable marker. DNA encoding a form of prM with a pr/M cleavage site mutation was used to suppress the cell-fusion activity of VLPs. After transfection with the resultant plasmid, Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were incubated with blasticidin, and cells resistant to the antibiotic were obtained. Western blot analysis and enzyme-linked immunosorbent assay of a culture supernatant showed that transfected High Five cells secreted an E antigen equivalent to the authentic JEV E. Sucrose density-gradient sedimentation analysis of the culture supernatant from recombinant High Five cells indicated that secreted E antigen molecules were produced in a particulate form. VLPs recovered from the supernatant successfully induced neutralizing antibodies in mice, particularly when adsorbed to alum adjuvant. High yields (≈30 μg/ml) of E antigen were achieved in shake-flask cultures. These results indicate that recombinant insect cells may offer a novel approach for efficient VLP production.     

Molecular cloning of giant panda pituitary prolactin cDNA and its expression in Escherichia coli

cDNA encoding pituitary (PRL) of giant panda was obtained using RT-PCR and expressed in E. coli. The results revealed that panda PRL cDNA encodes a precursor protein of 229 amino acids including a putative signal peptide of 30 amino acids and a mature protein of 199 residues with one potential N-glycosylation site. Sequence comparison indicated that panda PRL shares a high degree of identity to other known PRL sequences ranging from 98% with mink PRL to about 50% with rodent PRL. Six cysteine residues and 29 conserved residues distributed in four domains (PD1, PD2, PD3, and PD4) of PRL were observed. through multiple sequence alignment. Fourteen key residues of binding sites 1 and 2 involved in receptor binding are conserved in panda PRL. GST fused recombinant panda PRL protein was efficiently expressed with the form of insoluble inclusion bodies in E. coli BL21 transformed with a pGEX-4T-1 expression vector containing the DNA sequence encoding mature panda PRL. Western blot analysis indicated that GST-panda PRL recombinant protein could be recognized by antibody against human PRL. Our results would contribute to further elucidating the structural and functional characteristics of pituitary PRL and provide a basis for the production of recombinant panda prolactin for future use in the breeding of giant panda.