Construction of an Escherichia coli expression vector for the non-structural (NS)-1 protein of avian influenza virus H5N1

In the search for universal vaccine candidates for the prevention of avian influenza, the non-structural (NS)-1 protein 1 of avian influenza virus (AIV) H5N1 has shown promising potential for its ability to effectively stimulate the host immunity. This study was aimed to produce a bacterial expression plasmid using pRSET B vector to harbour the NS1 gene of AIV H5N1 (A/Chicken/Malaysia/5858/2004 (H5N1)) for protein expression in Escherichia coli (E. coli). The NS1 gene (687 bp) was initially amplified by polymerase chain reaction (PCR) and then cloned into a pGEM-T Easy TA vector. The NS1 gene was released from pGEM-T-NS1 using EcoRI and XhoI restriction enzymes (RE). The pRSET B vector was also linearized using the same RE. The digested NS1 gene and linearized pRSET B were ligated using T4 DNA ligase to form the expression plasmid, pRSET B-NS1. The NS1 gene sequence in pRSET B-NS1 was confirmed by DNA sequencing. To prepare recombinant bacterial cells for protein expression in the future, pRSET B-NS1 was transformed into E. coli strain BL21 (DE3) by heat-shock. Colonies bearing the recombinant plasmid were screened using PCR. The DNA sequencing analysis revealed that the NS1 gene sequence was 97% homologous to that of AIV H5N1 A/Chicken/Malaysia/5858/2004 (H5N1). These results indicated that the NS1 gene of influenza A/Chicken/Malaysia/5858/2004 (H5N1) was successfully amplified and cloned into a pRSET B vector. Bacterial colonies carrying pRSET B-NS1 can be used for the synthesis of NS1-based influenza vaccine in the future and thereby aid in the prevention of avian influenza

Replication of a Malaysian Strain Avian Influenza A Virus H5N1 in Madin-Darby Canine Kidney and African Green Monkey Kidney cells

The use of cell lines such as Madin-Darby Canine Kidney (MDCK) and African Green Monkey Kidney (Vero) cells in influenza vaccine production is much advocated presently as a safer alternative to chicken embryonated eggs. It is thus essential to understand the influenza virus replication patterns in these cell lines prior to utilizing them in vaccine production. The infectivity of avian influenza A virus (A/Chicken/Malaysia/5858/2004) H5N1 in MDCK and Vero cell lines was first assessed by comparing the cytopathic effect (CPE) caused by the virus infection. The viral loads in both of the infected media and cells were also compared. The results showed that both of the MDCK and Vero cells began to exhibit significant CPE (p<0.05) after 48 h post-infection (h p.i). The MDCK cell line was more susceptible to the virus infection compared to Vero cell line throughout the incubation period. A higher viral load was also detected in the host cells compared to their respective culturing media. Interestingly, after reaching its maximum titer at 48 h p.i, the viral load in MDCK cells declined meanwhile the viral load in Vero cells increased gradually and peaked at 120 h p.i. Overall, both cell lines support efficient H5N1 virus replication. While the peak viral loads measured in the two cell lines did not differ much, a more rapid replication was observed in the infected MDCK samples. The finding showed that MDCK cell line might serve as a more time-saving and cost-effective cell culture-based system compared to Vero cell line for influenza vaccine production

Purification of His-tagged hepatitis B core antigen from unclarified bacterial homogenate using immobilized metal affinity-expanded bed adsorption chromatography

Hepatitis B core antigen (HBcAg) is used as a diagnostic reagent for the detection of hepatitis B virus infection. In this study, immobilized metal affinity-expanded bed adsorption chromatography (IMA-EBAC) was employed to purify N-terminally His-tagged HBcAg from unclarified bacterial homogenate. Streamline Chelating was used as the adsorbent and the batch adsorption experiment showed that the optimal binding pH of His-tagged HBcAg was 8.0 with a binding capacity of 1.8 mg per ml of adsorbent. The optimal elution condition for the elution of His-tagged HBcAg from the adsorbent was at pH 7 in the presence of 500 mM imidazole and 1.5 M NaCl. The IMA-EBAC has successfully recovered 56% of His-tagged HBcAg from the unclarified E. coli homogenate with a purification factor of 3.64. Enzyme-linked immunosorbent assay (ELISA) showed that the antigenicity of the recovered His-tagged HBcAg was not affected throughout the IMA-EBAC purification process and electron microscopy revealed that the protein assembled into virus-like particles (VLP)

Evaluasi keselamatan Lactobacillus casei C1 pada tikus Wistar

Pada masa kini, probiotik diambil sebagai makanan tambahan secara meluas untuk kebaikan kesihatan. Namun begitu, masih kurang kajian keselamatan terhadap kebanyakan bakteria probiotik. Tambahan pula, khasiat probiotik amat bergantung pada strain yang digunakan dalam penghasilan produk probiotik. Oleh itu, kajian ini amat penting bagi mengkaji keselamatan pengambilan Lactobacillus casei (Lb. casei) C1 yang baru dipencilkan secara oral. Sebanyak 32 ekor tikus Wistar (WIS) digunakan dalam kajian ketoksikan oral akut (dos tunggal) dan subakut (dos berulangan selama 28 hari). Tikus dibahagikan kepada kumpulan kawalan yang diberi salin penimbal (PBS) dan kumpulan kajian yang diberi Lb. casei C1 (1011 CFU/ml) secara oral. Dalam ketoksikan oral akut, rawatan diberi sekali setiap 24 jam dan dipantau selama 14 hari. Untuk ketoksikan oral subakut, rawatan diberi setiap hari selama 28 hari dan kesan dipantau sepanjang tempoh masa kajian. Berat badan, makanan dan air direkodkan. Kumpulan akut dan subakut dikorbankan pada hari ke-15 dan ke-29. Serum dikumpul untuk menentukan aras protein jumlah, malondialdehid (MDA), alanina aminotransferase (ALT), aspartat aminotransferase (AST), laktat dihidrogenase (LDH) dan kreatinin. Organ pula diambil untuk pemerhatian histologi. Tiada perbezaan yang signifikan (p > 0.05) diperhatikan dalam berat badan, pengambilan makanan dan minuman antara tikus kawalan dan kajian dalam kumpulan ketoksikan oral akut. Bilangan sel darah, aras jumlah protein, MDA, LDH dan kreatinin juga tidak menunjukkan perbezaan yang signifikan antara tikus kawalan dengan kajian. Hasil yang sama juga direkod untuk kumpulan ketoksikan oral subakut kecuali aras ALT dan AST yang menunjukkan perbezaan signifikan (p < 0.05). Tiada perubahan morfologi yang ketara diperhatikan pada organ hepar, ginjal dan ileum tikus kajian berbanding dengan tikus kawalan dalam kedua-dua kumpulan kajian. Kesimpulannya, Lb. casei C1 tidak mempunyai kesan toksik pada tikus Wistar maka ia adalah selamat untuk diambil secara oral

Vaksin oral universal untuk jangkitan influenza

Saban tahun, jangkitan virus influenza A telah mengakibatkan kadar kematian yang setinggi 300,000-500,000 di seluruh dunia. Biarpun terdapat agen dan vaksin anti-influenza yang berkesan terhadap jangkitan influenza, namun kadar mutasi yang tinggi dalam virus influenza A telah menyebabkan keberkesanan agen atau vaksin tersebut menurun secara mendadak dalam sesetengah individu. Keadaan ini dirumitkan lagi dengan beberapa batasan dalam proses penghasilan vaksin influenza yang sedia ada, antaranya tempoh produksi yang lama, kapasiti vaksin yang terhad dan kekurangan perlindungan bersilang terhadap subjenis virus influenza A yang berlainan. Bagi menyelesaikan isu-isu tersebut, pembangunan vaksin influenza universal berdasarkan antigen yang terpelihara seperti protein bukan struktur 1 (NS1) telah diusahakan. Protein NS1 amat terpelihara dalam kesemua subjenis virus influenza A yang telah diketahui sehingga kini, banyak dihasilkan pada permukaan sel terjangkit dan berperanan penting untuk mengekalkan kevirulenan virus. Di samping itu, sel limfosit-T sitotoksik yang aktif terhadap NS1 juga mampu mencegah perebakan jangkitan influenza dalam perumah. Bagi menghalang penyebaran jangkitan influenza dengan lebih efektif, imunisasi secara oral telah lama dicadangkan kerana kaedah ini adalah lebih mudah serta selamat malahan dapat menyasarkan virus influenza A bermula dari laluan kemasukannya. Lactobacillus telah banyak dikaji peranannya sebagai pembawa bakteria dalam pembangunan vaksin oral disebabkan ciri-ciri probiotiknya yang signifikan. Antaranya, dapat merangsang tindak balas imun di lapisan mukosa oral dan saluran pernafasan, kolonisasi tinggi pada permukaan mukosa serta keadjuvanan semula jadi yang tinggi. Dengan itu, vaksin influenza universal oral yang dihasilkan dengan menggunakan NS1 dan Lactobacillus seharusnya dikaji secara terperinci lagi dalam reka bentuk vaksin oral influenza

An N-terminal extension to the hepatitis B virus core protein forms a poorly ordered trimeric spike in assembled virus-like particles

Virus-like particles composed of the core antigen of hepatitis B virus (HBcAg) have been shown to be an effective platform for the display of foreign epitopes in vaccine development. Heterologous sequences have been successfully inserted at both amino and carboxy termini as well as internally at the major immunodominant epitope. We used cryogenic electron microscopy (CryoEM) and three-dimensional image reconstruction to investigate the structure of VLPs assembled from an N-terminal extended HBcAg that contained a polyhistidine tag. The insert was seen to form a trimeric spike on the capsid surface that was poorly resolved, most likely owing to it being flexible. We hypothesise that the capacity of N-terminal inserts to form trimers may have application in the development of multivalent vaccines to trimeric antigens. Our analysis also highlights the value of tools for local resolution assessment in studies of partially disordered macromolecular assemblies by cryoEM

Cytotoxicity, regulation of apoptotic and anti-apoptotic gene expression by IL-27 in MCF-7 and MDA-MB-231 breast cancer cell lines

Breast cancer is one of the commonest cancers among women. Conventional therapies cause adverse side effects in patients. Cytokine immunotherapy such as interleukin-27 (IL-27) has been sought as an alternative cancer treatment in recent years. IL-27 has been shown to improve anticancer immunity and anti-angiogenesis in cancers, however, its effect on apoptotic and anti-apoptotic gene expression especially in breast cancers is yet to be explored. Cytotoxicity of IL-27 in non-cancerous (184b5) and cancerous (MCF-7 and MDA-MB-231) breast cell lines was first determined for 24-72 h in this study. The results indicated that IL-27 treatment did not retard 184b5 cell growth, however, did inhibit MCF-7 (48 h) and MDA-MB-231 (72 h) cell growth with IC50 at 442 and 457 ng/ml, respectively. Apoptotic (TRAIL, FADD, FAS, caspase-3 and caspase-8) and anti-apoptotic (BCL-2, AKT, and COX-2) genes were then amplified from untreated (control) and treated breast cancer cells and studied. TRAIL, caspase-3, caspase-8 gene expression was significantly (p < 0.05) upregulated in treated MCF-7 (442 ng/ml) and MDA-MB-231 (457 ng/ml) cells. Expression of FADD and FAS genes was not detected in both control and treated MCF-7 and MDA-MB-231 cells. COX-2 gene was also not expressed by MCF-7 cells, but reduced significantly (p < 0.05) in treated MDA-MB-231 cells. In MDA-MB-231 cells, IL-27 treatment seemed to slightly enhance the expression of AKT and BCL-2 genes which, on the other hand, was downregulated in treated MCF-7 cells. Conclusively, IL-27 is able to inhibit breast cancer cell growth and regulate apoptotic and anti-apoptotic gene expression in breast cancer cells

A systematic review on the effectiveness of enteral immunonutrition (EIN) on pre- and post-operative outcomes in gastric cancer patients

Gastric cancer is one of the most common upper gastrointestinal malignancies. To date, enteral immunonutrition (EIN) has gained increasing attention as it is found to effectively enhance the host’s immunity and improve the metabolic status of gastric cancer patients undergoing gastrectomy. The health-boosting effects of EIN are believed to originate from a number of nutritional elements such as omega-3 fatty acids, glutamine, arginine and nucleic acid precursors that help reduce the incidences of post-operative complications and shorten the length of hospital stay among the aforementioned patients. However, little was known about the consistency of health-boosting benefits conferred by EIN. Hence, according to PRISMA (Preferred Reporting Items for Systematic reviews and Meta-Analyses) protocol, this systematic review was carried out using nine meticulously and specifically selected full-length articles focusing on the pre- and post-operative effects of EIN including physical, biochemical, clinical and immunological outcomes on gastric cancer patients. Among the selected articles, seven of them focused on post-operative EIN while the remaining two concentrated on pre-operative EIN. In most of the selected studies, more than one immunonutritional components (arginine, glutamine, omega-3 fatty acid, RNA) were integrated. Patients receiving EIN showed significantly improved immunity, for example, increase in CD4+ T and NK cell counts that are responsible for fighting pathogens. In addition to that, individuals receiving EIN also showed increased levels of inflammatory biomarkers in their sera such as pre-albumin and transferrin. This results in shorter period of post-operative hospital stay that in turn permits progressive healing process and increases the survival rate due to minimal frequency of post-operative infections. Conclusively, our systematic review acknowledges that regardless of the initiation timing (pre-operative or post-operative) of immunonutrition, EIN can improve the overall health status of gastric cancer patients including infection complications and the length of hospital stay through regulation of immune responses

Mutagenicity and antimutagenic activities of lactic acid bacteria (LAB) isolated from fermented durian (tempoyak)

Mutagenic and antimutagenic activities of lactic acid bacteria (LAB) Lactobacillus plantarum isolated from the local fermented durian (tempoyak) was determined by Ames test (Salmonella/microsome mutagenicity assay). Our study also involved pre-incubation assay against Salmonella typhimurium TA 98 and TA 100 bacterial strain in the presence and absence of metabolic activator S9 system. It was found that the L. plantarum showed no mutagenic activity on both S. typhimurium strain TA 98 and TA 100 in the presence and absence of metabolic activator. Significant antimutagenic activity (p < 0.05) was observed in both cell-free supernatant and bacterial cell suspension of L. plantarum as compared to the mutagenicity induced by 2-Aminoanthracene in the presence of metabolic activator. Meanwhile, in the absence of metabolic activator, only the bacterial cells of L. plantarum showed antimutagenicity acitivity against Sodium Azide and 2-Nitrofluorene. In conclusion, L. plantarum could play a vital role as chemopreventive agent by binding to mutagens and suppressing mutagenesis. Thus, L. plantarum could be consider as a good candidate for functional food development as a supplement product to prevent development of colon cancer

Display of the antigenic region of Nipah virus nucleocapsid protein on hepatitis B virus capsid

The C-terminal domain of Nipah virus (NiV) nucleocapsid protein (NP 401-532) was inserted at the N-terminus and the immunodominant loop of hepatitis B core antigen (HBc). The stability of NP 401-532 increased tremendously when displayed on the HBc particles. These particles reacted specifically with the swine anti-NiV and the human anti-HBc antisera