CONSECUTIVE IMMUNIZATION WITH RECOMBINANT FOWLPOX VIRUS AND PLASMID DNA FOR ENHANCING CELLULAR AND HUMORAL IMMUNITY

Objective: To investigate the influence of consecutive immunization on cellular and humoral immunity in mice. Methods: We evaluated a consecutive immunization strategy of priming with recombinant fowlpox virus vUTALG and boosting with plasmid DNA pcDNAG encoding HIV-1 capsid protein Gag. Results: In immunized mice, the number of CD 4 + T cells from splenic lymphocytes increased significantly and the proliferation response of splenocytes to ConA and LPS elevated markedly and HIV-1-specific antibody response could be induced. Conclusion: Consecutive immunization could increase cellular and humoral immunity responses in mice.

Construction and Expression of Recombinant Plasmid pCD-rbFGF in Osteoblasts

Summary: To construct basic fibroblast growth factor (bFGF) eukaryotic expression vector and to evaluate the possibility of bFGF gene therapy in orthopedic disease, the pCD-rbFGF recombinant plasmid was constructed by cloning rat basic fibroblast growth factor (bFGF) cDNA into an eukaryotic expression vector, pcDNA 3. Rat osteoblasts were transfected with pCD-rbFGF plasmid by lopofectin mediated gene transfer, the transient expression was detected by streptavidin-biotin-enzyme complex (SABC) method. It was observed that the expression of rat bFGF gene was detected 72 h after transfected distinctly. Basic fibroblast growth factor gene therapy is a method of potential for a wide array of orthopedic diseases.

Construction and Expression of Recombinant Ghrelin Plasmid and Effects on Growth Performance and Gastric Acid Secretion of Rats

The paper was to study construction and expression of rGhrelin （pcDNA3 -Ghrelin） and its effect on growth performance and gastric acid secretion of rats. Ghrelin amplified from gastric mucosa of weaned piglets was cloned into the expression vector pcDNA3 to get recombinant plasmid pcDNA3 - Ghrelin. Twelve weaning rats were randomly divided into two groups, six rats each group. The rats in each treatment group were individually injected with 100pg of naked plasmid pcDNA3 -Ghrelin, and the rats in control group were injected with empty plasmid. The weights and feed consumption of rats were measured after injection for 7, 14 and 29 d, respectively. The rats were sacrificed at the end of the experiment, and their stomach was separated and weighed, the pH value of gastric juice was measured as well. The results showed that the average daily gain of rats at 7 and 29 d were significantly higher than that in control group, respectively （P〈0.05）, and feed consumption did not have significant chan- ges; the feed meat ratio of rats in the treatment groups was significantly lower than that in control group （ P 〈0.05） ; the gastric relative weight and gastric weight did not change significantly, while the pH value of gastric juice of rats in treatment groups was significantly lower than that in control group （P 〈 0.05）. This indicated that after transfected expression of muscle tissue, ghrelin played an important regulatory role in growth and gastric acid secretion of rats.

Detection of the copy number of plasmid encoding HBsAg in recombinant yeast by PCR relative quantitative assay

The purpose of this study is to detect the plasmid copies encoding HBsAg in recombinant yeast Saccharomyces cerevisiae by real-time PCR, and provide the data for setting up an optimal process of manufacturing hepatitis B vaccine in future. A single copy URA3 gene in the yeast genome was employed as internal control to test the plasmid copies by real-time PCR. The yeast cells carrying plasmids encoding HBsAg were cultured in regular non-feeding process and fed-batch process in shaking flasks at laboratory level and industrial fermentor. The cells at different stages of cultivation were collected and broken. Then the total DNAs of the cells were extracted and the plasmid copies were detected by real-time PCR with the method of relative quantification. The method of the real-time relative quantitative PCR used in this study was reliable with good accuracy and quickness. The plasmid copies in non-feeding and fed-batch flasks were 39.22±12.00 and 43.06±5.70, while the copies in fermentor were 96.16±21.00 and 82.50±7.78, respectively. The data show that the variation of the result is very small and the data precision is quite good. To prolong the fermentation time may increase the cell density without influence on the stability of the plasmid, and the method of relative quantification can also be applied to test the copy number of interest target gene in any host cell with certain known copy gene.

A novel recombinant DNA vaccine encodingMycobacterium tuberculosis ESAT-6 and FL protects againstMycobacterium tuberculosis challenge in mice

Mycobacterium tuberculosis 6-kDa early secretory antigenic target （ESAT-6） is a dominant target antigen for cell-mediated immunity in the early phase of tuberculosis. The fms-like tyrosine kinase 3 ligand （FL） that induces potent immune response has been used as an adjuvant in vaccine development. In this study, a new recombinant plasmid （plRES-epitope-peptides-FL） encoding three T cell epitopes of ESAT-6 and FL was constructed, and the immunogenicity of the DNA vaccine was assessed in C57BL/6 mice immunized with the plasmid DNA vaccine. Additionally, a strategy of intramuscular injection with the DNA vaccine （prime） and intranasal administration of the epitope peptides （boost） was employed to induce higher immune reaction of the mice. The results showed that mice vaccinated with the recombinant plasmid DNA vaccine and boosted with the peptides not only increased the levels of Thl cytokines （IFN-γ and IL-12）, the number of IFN-γ＋ T cells and activities of cytotoxic T lymphocytes as well as IgG, but also enhanced protection against Mycobacterium tuberculosis challenge. In conclusion, these data indicate that the novel recombinant plRES-epitope-peptides-FL plasmid is a useful DNA vaccine for pre- venting Mycobacterium tuberculosis infection.

The expression and antigenicity identification of recombinant rat TGF-β1 in bacteria

In order to study structure-function details of TGF-beta1, the recombinant mature form of rat TGF-beta1 was expressed in bacteria. Synthesis of the 112 amino-acid carboxyl-terminal part of TGF-beta1 (amino acid 279-390) was controlled by an inducible gene expression system based on bacteriophage T7 RNA polymerase. This system allowed an active and selective synthesis of recombinant TGF-beta1. The molecular weight of expressed TGF-alpha1 monomer determined on SDS-polyacrylamide gel under reducing conditions was about 13 kD. Serial detergent washes combined with a single gel-filtration purification step were sufficient to purify the expression product to homogeneity. Amino-terminal sequencing revealed that the N-terminal of the recombinant protein was identical to the published data. In Western blot analysis the recombinant polypeptide showed excellent antigenicity against polyclonal TGF-beta1 antibody. The mature recombinant rat TGF-beta1 expressed in this study provides a useful tool for future detailed structural and functional studies.

Study on DNA Immunization by Recombinants Encoding Japanese Encephalitis Virus prME and E Proteins

To study the expression characteristic of Japanese encephalitis virus (JEV) prME and E proteins and the efficacy of DNA immunization by different recombinant plasmids containing JEV prME (2001 bp) and E (1500 bp) genes, two recombinants (pJME and pJE) containing JEV prME and E genes fused with FLAG were constructed and then transfected into HepG2 and COS-1 cells by liposome fusion. The expression feature of FLAG-prME (about 72 kDa) and FLAG-E (about 54 kDa) proteins in transfected cells were analyzed by Western blot and two antibody systems (anti-FLAG and anti-E). BALB/c mice were immunized with 100 μg of two kinds of recombinants by intramuscular injection, and JEV JaGAr-01 strains (10 5 PFU/100 μl)were given to BALB/c mice by intraperioneal injection 3 wk after twice DNA immunization by a lethal virus challenge. BALB/c mice were observed for 21 days after challenge. 80% plaque reduction neutralization test was performed to titrate neutralization antibody before and after viral challenge. It was found that the expression of proteins associated with pJME and pJE was determined in transfected cells with anti-FLAG and a new protein of 11 kDa was detected in HepG2 and COS-1 cells transfected with pJME. Only E (53 kDa) protein was identified as transfected with pJME using anti-E. Higher level of neutralization antibodies and the efficacy of protective immunity were induced with pJME immunization, and were similar to those induced by inactivated Japanese encephalitis vaccine, but were better than those induced with pJE. It concludes that the expression level from prM to E proteins of JEV is different in vitro, and the in vitro expression efficiency of pJME was better than that of pJE. FLAG-prME protein expressed by pJME could be cleaved by peptidase from host. The efficacy of DNA immunization is correlated to the expression characterization of related proteins expressed in vitro.

表达SFTSV Gn基因的重组5型腺病毒的构建与鉴定

为了构建出表达新布尼亚病毒(SFTSV)Gn基因的重组5型腺病毒,试验根据GenBank上发表的SFTSV Gn基因序列(登录号为ADZ04482.1)设计合成引物,采用特异性PCR方法在Gn基因前添加了KOZAK序列和tPA信号肽序列;然后通过酶切、连接等方法构建重组穿梭质粒PGA-KOZAK-tPA-Gn,与腺病毒骨架质粒pAd5-ΔE1ΔE3-5E4在BJ5183感受态细胞中进行同源重组,得到重组腺病毒质粒rAd5-KOZAK-tPA-Gn,用PacⅠ酶酶切重组腺病毒质粒,并将线性化的质粒转染至HEK 293细胞中进行病毒包装、扩繁和纯化;采用PCR扩增、Western-blot等技术检测病毒基因的表达情况,并测定重组腺病毒效价。结果表明:通过PCR扩增获得了1 546 bp的KOZAK-tPA-Gn基因,并成功构建了重组穿梭质粒;SFTSV Gn基因在重组腺病毒传代过程中稳定存在;重组腺病毒在HEK 293细胞中表达出分子量约为61 ku的SFTSV Gn蛋白;测得重组腺病毒效价为1×10^(-7.63)/0.1 mL TCID_(50)。说明试验成功构建出表达SFTSV Gn基因的重组5型腺病毒。

小鼠内皮抑素基因克隆、测序及pEgr-IFNγ-mEndostatin重组双基因表达质粒的构建

目的 :克隆小鼠内皮抑素 (m Endostatin)编码区 c DNA序列并构建含 Egr- 1启动子的 IFNγ和 m Endo-statin双基因表达载体。方法 :利用逆转录多聚酶链反应法 (RT- PCR) ,以小鼠肝细胞 m RNA为模板 ,扩增获得全长 m Endostatin,与 p MD1 8T载体连接作全自动测序 ,并利用基因重组技术构建含 Egr- 1启动子的 IFNγ和m Endostatin双基因表达质粒。结果 :经测序证实获得的 m Endostatin序列与文献报道完全一致 ,并构建了含 Egr-1启动子的 IFNγ和 m Endostatin双基因表达质粒 p Egr- IFNγ- m Endostatin。结论 :利用 RT- PCR法成功克隆了m Endostatin的 c DNA序列 ,构建了 p Egr- IFNγ- m Endostatin重组双基因表达质粒。

丙二酸盐克罗诺杆菌rpoS基因突变株与回补株的构建

丙二酸盐克罗诺杆菌(Cronobacter malonaticus)在面对环境胁迫时,通过自身调节机制适应压力。rpoS基因作为一种稳定期σ因子和压力应答过程中的主要调节因子,在细菌抵抗或耐受环境胁迫压力中发挥重要作用。文章运用red同源重组技术和质粒表达系统,构建C.malonaticus G362的rpoS基因缺失株ΔrpoS和回补菌株ΔrpoS-com,为研究丙二酸盐克罗诺杆菌中rpoS基因的功能提供遗传材料。生长曲线测定结果表明,rpoS基因缺失未对正常培养条件下细菌生长产生显著影响。

SARS-CoV-2 M蛋白生物信息学分析及真核表达载体的构建与鉴定

目的利用生物信息学方法预测新型冠状病毒膜(SARS-CoV-2 M)蛋白的结构与性质,构建SARS-CoV-2 M蛋白真核表达载体,为抗新型冠状病毒药物和疫苗的研发提供一定参考。方法利用NCBI查找SARS-CoV-2 M蛋白的核苷酸序列和氨基酸序列,利用ORF Finder预测SARS-CoV-2 M蛋白的开放阅读框数目;Protparam预测SARS-CoV-2 M蛋白的理化性质;SignalP和TMHMM2.0预测SARS-CoV-2 M蛋白的信号肽与跨膜区;NetPhos、SOPMA和SWISS MODEL预测SARS-CoV-2 M蛋白的磷酸化位点、糖基化位点、二级结构与三级结构;IEDB、Immunomedicine Group、Cell-Ploc 2.0和Uniprot预测分析SARS-CoV-2 M蛋白的T细胞和B细胞表位、抗原决定簇、亚细胞定位及与其相互作用的蛋白。通过合成目的基因后插入到真核表达载体pcDNA3.1(+)中,双酶切鉴定后进行序列测序,将重组质粒转染至Vero细胞中,再以Western blot方法检测该基因编码蛋白的表达情况。结果编码SARS-CoV-2 M蛋白基因全长669 bp,由222个氨基酸组成;其分子式为C1165H1823N303O301S8,相对分子质量为25146.62,理论等电点为9.51,不稳定系数为39.14,脂肪族氨基酸系数为120.86,平均亲水系数为0.446;SARS-CoV-2 M蛋白有5个开放阅读框,无信号肽,3个跨膜区,15个磷酸化位点,1个糖基化位点,二级结构以无规则卷曲为主,占比45.5%;6个B细胞表位,8个Th细胞表位,平均抗原倾向为1.0532,主要与11个蛋白发生相互作用。成功构建了pcDNA3.1(+)-M重组质粒,双酶切鉴定插入片段大小正确,DNA测序结果与SARS-CoV-2 M基因序列同源性为100%,Western blot成功检测出该蛋白在Vero细胞内的表达。结论成功预测了SARS-CoV-2 M蛋白多项生物信息并成功构建pcDNA3.1(+)-M重组质粒,为抗新型冠状病毒药物和疫苗的研发提供了一定参考。

SOX7基因慢病毒载体的构建及其在缺氧条件下对HUVECs增殖与迁移功能的影响

目的构建过表达SRY-box transcription factor 7(SOX7)基因的慢病毒载体,建立SOX7基因过表达的人脐静脉内皮细胞株(Human umbilical vein endothelial cells,HUVECs),并在常氧和缺氧条件下探究过表达SOX7对HUVECs迁移和增殖能力的影响。方法使用聚合酶链式反应(Polymerase chain reaction,PCR)技术扩增SOX7基因,通过同源重组法构建以pHAGE-CMV-MCS-IRES-ZsGreen为载体的SOX7-pHAGE重组质粒。实时荧光定量PCR(Quantitative real-time PCR,qPCR)和免疫印迹(Western blot)法检测目的基因的mRNA和蛋白表达。将SOX7-pHAGE重组质粒和包装质粒共转染293T细胞,获得含有SOX7-pHAGE慢病毒悬液,经浓缩后感染HUVECs,构建SOX7稳定感染细胞株。应用qPCR和Western blot技术检测SOX7基因的mRNA和蛋白的表达。在常氧及缺氧条件下,通过细胞划痕实验和CCK-8法检测过表达SOX7对HUVECs迁移和增殖能力的影响。结果SOX7-pHAGE重组质粒经测序后,序列比对正确。qPCR和Western blot法成功检测出SOX7-pHAGE重组质粒转染的293T细胞在mRNA水平过表达约111.4倍(P=0.0028),在蛋白质水平上出现过表达条带;qPCR和Western blot技术成功检测到SOX7-pHAGE慢病毒感染的HUVECs在mRNA水平上显著升高约68.2倍(P=0.0030),在蛋白水平上显著升高约1.7倍(P=0.0043),SOX7-pHAGE重组质粒及SOX7稳定感染细胞株构建成功。在常氧条件下,SOX7稳定感染细胞株的迁移能力没有发生改变(P>0.9999),而在缺氧条件下其迁移能力增强约1.2倍(P=0.0044)。在常氧条件下,SOX7稳定感染细胞株的增殖能力增强约1.3倍(P=0.0087),在缺氧条件下其增殖能力也显著增强约1.4倍(P=0.0228)。结论SOX7稳定感染细胞株可促进HUVECs的增殖能力。在缺氧条件下,SOX7稳定感染细胞株可回补HUVECs被抑制的迁移和增殖能力。

人C/EBP β结构与功能的生物信息学分析及原核表达

目的 研究人CCAAT/增强子结合蛋白β(CCAAT/enhancer binding proteins, C/EBP β)原核表达载体的构建和蛋白表达,并通过生物信息学分析其结构和生物学功能。方法 收集对数生长期的肝癌细胞SMMC-7721,通过TRIzol法提取SMMC-7721细胞内总RNA,并以RNA为模板采用RT-PCR获得C/EBP β基因的编码序列,通过同源重组的方法将目的基因与原核表达质粒pET-28a(+)相连接,经过大肠杆菌转化、抗性筛选、质粒提取、酶切和DNA测序获得重组质粒pET-28a(+)-C/EBP β。将重组质粒导入大肠杆菌BL21中,通过异丙基-β-D-硫代半乳糖苷(isopropyl β-D-1-thiogal, IPTG)诱导C/EBP β重组蛋白表达,采用镍离子亲和层析法纯化C/EBP β融合蛋白,通过蛋白免疫印迹验证目的蛋白并分析C/EBP β的蛋白纯度。同时对人C/EBP β编码的蛋白质进行理化性质、亲水/疏水性、二级结构以及磷酸化位点等生物信息学分析。结果 通过RT-PCR成功获得人C/EBP β基因,构建的pET-28a(+)-C/EBP β重组质粒经测序鉴定C/EBP β DNA序列完全正确,表明重组pET-28a(+)-C/EBP β质粒构建成功。C/EBP β蛋白是一个性质不稳定的亲水蛋白质,由345个氨基酸组成,分子量为36.105 kD,等电点为8.55。其是一种胞内蛋白质,主要分布在细胞质和细胞核。对其结构分析发现:无规则卷曲是其主要的二级结构,为60.87%。蛋白免疫印迹结果显示通过亲和层析纯化后获得较高纯度的C/EBP β。结论 本实验成功克隆并构建人pET-28a(+)-C/EBP β重组质粒,获得较高纯度的C/EBP β,为进一步C/EBP β蛋白功能的研究和抗体的制备奠定了良好的基础。

Expression,purification and immunocharacteristics of recombination UreB protein of H.pylori

INTRODUCTIONHelicobacter pylori (H . pylori) is associated with the development of chronic gastritis ,peptic ulcer and gastric cancer and gastric MALT lymphoma[1-9],H .pylori has many antigens ,including urease ,heat shock protein and vacuolating cytotoxin and so on ,and urease is an important factor in the colinization of the gastric mucosa and suspected to cause damage to the gastric mucosa[10-14].At the same time ,urdase is also one of the important protective antigens .

Construction of Prokaryotic Expression Plasmid of Fusion Protein Including Porin A and Porin B of Neisseria Gonorrhoeae and Its Expression in E.coli

In order to provide a rational research basis for clinical detection and genetic engineering vaccine, plasmid pET-28a (+) encoding both Porin gene PIA and PIB of Neisseria gonorrhoeae was constructed and a fusion protein in E.coli DE3 expressed. The fragments of PIA and PIB gene of Neisseria gonorrhoeae were amplified and cloned into prokaryotic expression plasmid pET-28a(+) with double restriction endonuclease cut to construct recombinant pET-PIB-PIA. The recombinant was verified with restriction endonuclease and sequenced and transformed into E.coli DE3 to express the fusion protein PIB-PIA after induced with IPTG. The results showed PIA-PIB fusion DNA fragment was proved correct through sequencing. A 67 kD (1 kD=0 992 1 ku) fusion protein had been detected by SDS-PAGE. It was concluded that the fusion protein was successively expressed.

GI和GII诺如病毒ERA的可视化快速检测

诺如病毒(NoV)是常见的食源性病毒之一,只要极少数量的NoV就可以导致机体感染发病,但目前还没有特效的治疗药物和疫苗,因此建立快速检测方法对食品中的NoV进行早期筛查至关重要。本研究首先通过装甲RNA技术将GB 4789.42规定的GI和GII NoV的靶标基因包裹到MS2噬菌体的衣壳蛋白中,制成内含GI和GII NoV两联检测靶标的重组质粒参考样品,其次基于酶促等温扩增(ERA)技术,分别设计了GI和GII NoV基础型ERA和荧光型ERA检测的引物和探针,并通过试验筛选确定了最佳的引物探针组合。从而建立了GI和GII NoV检测ERA显色法和荧光法两种可视化方法,通过肉眼可直接观测结果。进一步对反应程序进行了优化,将ERA显色法和荧光法的扩增时间分别缩短至5min和8min。并通过对反应体系的优化,将体系减半,从而降低了检测成本。在此情况下,对核酸参考样品检测的最低灵敏度分别可达10^(-2)、10^(-3)ng/μL。最后将本研究建立的可视化快速检测方法应用于真实的GI和GIINoV粪便样本检测,并对方法的性能参数进行分析,结果显示:建立的GI和GII NoV ERA可视化快速检测方法特异性良好,对其他食源性病毒无交叉扩增,最低可检出10拷贝/μL的NoV,可以满足GI和GII NoV可视化快速筛查的需求。本研究方法的建立为NoV的快速筛查和风险监测提供了良好的技术支撑,对于控制NoV爆发、保障人民健康具有重要意义。

耐热性α-半乳糖苷酶突变体的筛选及酶学性质研究

研究旨在改造株链球菌来源的α-半乳糖苷酶基因Gal_(2)7A,获得耐热性高的突变体。在常规PCR体系中加入不同水平的MnCl_(2),将基因克隆至载体pGAPZαA并成功构建表达酶产物的重组质粒pGAPZαA-Gal_(2)7A。将线性化的重组质粒电转至毕赤酵母GS115,在含有100 g/L博来霉素的YPDS平板上筛选耐热性提高的突变体,通过pNPG的方法测定重组酶的酶学性质为最适温度65℃,最适pH值6.5,Ag^(2+)、Hg^(2+)对重组酶的酶学活性具有明显的抑制作用,Cu^(2+)对重组酶的酶活性具有促进作用。研究表明,试验成功构建耐热α-半乳糖苷酶高效表达菌株29和54,在55℃保温10 min与同等条件下的野生型进行对比,加热前后的保留酶活比的增幅大于45%。

Construction of a recombinant plasmid harbouring the rhoptry protein 1 gene of Toxoplasma gondii and preliminary observations on DNA immunity

Objective To observe the immune responses elicited in BALB/c mice by a DNA vaccine. A gene encoding rhoptry protein 1 (ROP1) from Toxoplasma gondii (T. gondii) was cloned into vector pcDNA3. Methods Amplifyied gene fragments coding for ROP1 from the genomic DNA of T.gondii ZS2 were inserted into cloning vector, pUC18, and sub-cloned into pcDNA3. Mice were injected at a dosage of 100?μg recombinant plasmid DNA by intramuscular injection and boosted after 2 weeks. pcDNA3 and normal saline were used as control. 30, 50 and 70 days after the second immunization, NK cell activity, T lymphocyte proliferation and sub-clusters and serum IgG antibody were assayed.Results The specific gene fragment coding for ROP1 was amplified and a pcROP1 recombinant was constructed. At 30 days after immunization, the spleens of the mice were obviously enlarged evidently. NKC activity and the proliferation of spleen T lymphocytes seen on MTT assay were higher in pcROP1 group than in the controls. The number of CD4+ T cells exhibited no obvious increase compared with that of the control, but CD8+ T cells were obviously increased (P<0.05). At 90 days after vaccination, the titer of IgG antibody in the serum of vaccinated mice was positive (1∶100). Conclusion pcROP1 was constructed and it could elicit both cellular and humoral immune responses in immunized mice.

基于酿酒酵母的多片段质粒的构建

为避免现有的多片段质粒构建技术的弊端,提高多片段质粒构建效率,以构建树干毕赤酵母Agglutinin-like基因的敲除载体为例,利用酿酒酵母活体细胞重组系统,一次性将多个外源DNA片段和线性化质粒重组,形成环状质粒。通过引物设计在相互连接的DNA片段之间引入50bp重叠序列,用PCR扩增DNA片段后,与线性化质粒一起转化酿酒酵母,再用PCR和测序等方法鉴定阳性酵母菌落中质粒的正确性。通过本方法构建的多片段质粒正确率高、耗时短。

牛妊娠相关糖蛋白18重组载体构建、表达条件优化和生物信息学分析

为了制备牛妊娠相关糖蛋白18(boPAG18),并预测其理化性质和功能,本试验优化密码子并合成boPAG 18基因,构建pET30a-boPAG18重组质粒。以聚丙烯酰胺凝胶电泳(SDS-PAGE)测定的蛋白质表达量为指标,优化装液量、诱导剂浓度、诱导时间和诱导温度条件,采用亲和层析法纯化boPAG18,SDS-PAGE和免疫印迹法鉴定。预测boPAG18的疏水性、信号肽、修饰位点、结构和蛋白相互作用。结果显示,经双酶切鉴定,pET30a-boPAG18重组质粒构建成功,boPAG 18基因1143 bp,表达蛋白约38 kDa。确定最佳表达条件为在OD 600 nm值为0.6,装液量为40 mL,IPTG终浓度为0.2 g/L,37℃诱导4 h。纯化的boPAG18纯度高达98%,浓度为2.63 mg/mL。该蛋白分子式为C_(1923)H_(3007)N_(519)O_(541)S_(17),分子质量为42.6 ku,等电点为9.31,亲水性平均值为-0.041,为两性蛋白,蛋白质不稳定指数为42.41,在水中不稳定;脂肪系数为88.43,有381个氨基酸;消光系数为47370 mol-1·cm-1,半衰期为30 h。boPAG18前15个氨基酸为信号肽,无跨膜区域;有糖基化修饰位点57个,磷酸化修饰位点68个,B细胞抗原表位13个,二级结构中α-螺旋占比19.42%、β-转角占比5.77%、无规则卷曲占比44.88%、延伸链占比29.92%;有7个相互作用蛋白,可能参与了母体在妊娠期的泌乳、抗菌和神经保护等调节功能。本试验为深入研究boPAG18的结构和功能提供了数据支持。