The production of SPusp45-MSP-119 gene construct and its recombinant protein in Lactococcus lactis to be used as a malaria vaccine

Amino V.A. Kusuma, Apon Z. Mustopa, Wike Z. Mustafawi, Suharsono Suharsono



Background: Merozoite surface protein 1 (MSP-1) is a major protein used by the Plasmodium during red blood cells invasion in malaria. MSP-119, one of MSP-1 is highly conserved, and  it is a potential malaria vaccine candidate because the monoclonal antibodies are capable blocking erythrocyte invasion in vitro. The aim of this study was to produce MSP-119 gene construct and the recombinant protein in Lactococcus lactis.

Methods: Usp45-MSP-119, derived from codon optimization and the synthetic gene, was inserted into the pMAT cloning vector. A vector expressing MSP-119 included usp45 has been constructed by the manipulation of recombinant DNA using restriction enzymes. The MSP-119 protein was expressed to 45% ammonium sulfate precipitation and purified using Sephadex-G50 gel filtration chromatography. The expressed protein was characterized by SDS-PAGE and dot blot.

Results: usp45-MSP-119 gene was amplified using specific primers and inserted into the multiple cloning sites in the expression vector pNZ8148 with size 3,538 bp as a recombinant vector. The protein of  MSP-119 was successfully expressed in L. lactis with molecular weight of 10.45 kDa. The dot blot was tested in 3 different comparisons between the host cells, non-induced cells, and induced cells with 10 ng/ml nisin. The results showed that 10 ng/ml nisin gave a positive reaction as detected by dot blot assay.

Conclusion: This study confirmed that the usp45-MSP-119 gene was successfully inserted into the multiple cloning sites of the pNZ8148 expression vector and the MSP-119 protein expressed in the NICE system of the L. lactis host cell.


Lactococcus lactis; malaria; merozoite surface protein 1; nisin; usp45-MSP-119

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  1. [Internet]. World Health Organization Malaria Fact Sheet 2011. Geneva: WHO Media Centre. [updated 2011, cited 2015 May]. Available from:
  2. Kemenkes RI. Pusat Data Informasi Kesehatan: Profil Kesehatan Indonesia. Jakarta: Kementerian Kesehatan Republik Indonesia; 2011. Indonesia.
  3. Cui L, Mharakurwa S, Ndiaye D, Rathod PK, Rosenthal PJ. Antimalarial drug resistance: literature review and activities and findings of the ICEMR network. Am J Trop Med Hyg. 2015;93(3):57–68.
  4. Lazaraou M, Patino JAG, Jennings RM, Mclntosh RS, Shi J, Howell S, et al. Inhibition of erythrocyte invasion and Plasmodium falciparum merozoite surface protein 1 processing by human immunoglobulin G1 (IgG1) and IgG3 antibodies. Infect Immun. 2009; 77(12):5659–67.
  5. Baldwin MR, Li X, Hanada T, Lui S-C, Chisthi AT. Merozoite surface protein 1 recognition of host glycophorin A mediates malaria parasite invasion of red blood cells. Blood. 2015;125(17):2704–11.
  6. Cowman AF, Berry D, Baum J. The cellular and molecular basis for malaria parasite invasion of the human red blood cell. J Cell Biol. 2012; 198(6):961–71.
  7. Zhang ZH, Jiang PH, Li NJ, Shi M, Huang W. Oral vaccination of mice against rodent malaria with recombinant Lactococcus lactis expressing MSP-119. World J Gastroenterol. 2005;11(44):6975–80.
  8. Curd RD, Birdsall B, Kadekoppala M, Ogun SA, Kelly G, Holder AA. The structure of Plasmodium yoelli merozoite surface protein 119, antibody specificity and implications for malaria vaccine design. Open Biol. 2014;4:130091.
  9. Cruz-Gallardo I, Diaz-Moreno I, Diaz-Quintana A, Donaire A, Velazquez-Campoy A, Curd RD, et al. Antimalarial activity of cupredoxins: the interaction of Plasmodium merozoite surface protein 119 (MSP119) and rusticyanin. J Biol Chem. 2013; 288(9):20896-907.
  10. Ng DTW, Sarkar CA. Engineering signal peptides for enhanced protein secretion from Lactococcus lactis. Appl Environ Microbiol. 2013;79(1):347-56.
  11. Green MR, Sambrook J. Molecular Cloning: A Laboratory Manual. 4th ed. New York (US): Cold Spring Harbor Laboratory Press; 2012.
  12. Duan K, Dunn NW, Kim WS. Rapid plasmid DNA isolation from Lactococcus lactis using overnight cultures. Biotechnol Tech. 1999;13:519–21.
  13. Wu C, Zhang J, Du G, Chen J. Heterologous expression of Lactobacillus casei RecO improved the multiple-stress tolerance and lactic acid production in Lactococcus lactis NZ9000 during salt stress. Bioresour Technol. 2013;143:238–41.
  14. Todorov SD, Ho P, Vaz-Velho M, Dicks LMT. Characterization of bacteriocins produced by two strains of Lactobacillus plantarum isolated from Beloura and Chouriço, traditional pork products from Portugal. Meat Sci. 2010;84(3):334–43.
  15. Pertiwi W, Sartono TR, Sumarno, Adi S. Sensitivitas dan spesifisitas metode dot blot menggunakan antigen outer membrane protein Klebsiella pneumoniae yang direspon secretory-immunoglobulin A sputum penderita terinfeksi Klebsiella pneumoniae. J Respir Indones. 2009;29(3):1–15. Indonesian.
  16. Widjiati, Pradipta AR, Nazar DS, Estoepangestie ATS. Uji spesifisitas dengan dot blotting terhadap epidermal growth factor (EGF) yang diisolasi dari oosit kumulus komplek sapi setelah dimaturasi secara in vitro. Vet Med. 2014;7(2):134–9. Indonesian.
  17. Rattanachaikunsopon P, Phumkhachorn P. Glass bead transformation method for gram-positive bacteria. Braz J Microbiol. 2009;40(4):923–6.
  18. Heravi RM, Nasiraii R, Sankian M, Kermanshahi H, Varasteh AR. Optimization and comparison of two electrotransformation methods for Lactobacilli. Biotechnology 2012;11(1):50–4.
  19. De Ruyter PGGA, Kuipers OP, de Vos WM. Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin. Appl Environ Microbiol. 1996;62(10):3662–7.
  20. Korkmaz G, Holm M, Wiens T, Sanyal S. Comprehensive analysis of stop codon usage in bacteria and its correlation with release factor abundance. J Biol Chem. 2014;289(44):30334–42.
  21. Mohseni AH, Razavilar V, Keyvani H, Razavi MR, Khavari-Nejad RA. Efficient production and optimization of E7 oncoprotein from Iranian human papillomavirus type 16 in Lactococcus lactis using nisin-controlled gene expression (NICE) system. Microb Pathog. 2017;110:554–60.
  22. Wang ZH, Wang YL, Zeng XY. Construction and expression of a heterologous protein in Lactococcus lactis by using the nisin-controlled gene expression system: the case of the PRRSV ORF6 gene. Genet Mol Res. 2014;13(1):1088–96.
  23. Lages AC, Mustopa AZ, Sukmarini L, Suharsono. Cloning and expression of plantaricin w produced by Lactobacillus plantarum U19 Isolate from "Tempoyak" Indonesian fermented food as immunity protein in Lactococcus lactis. Appl Biochem Biotechnol. 2015;177:909–22.
  24. Mustopa AZ, Murtiyaningsih H, Fatimah, Suharsono. Cloning and heterologous expression of extracellular Plantaricin F produced by Lactobacillus plantarum S34 isolated from "Bekasam" in Lactococcus lactis. Microbiol Indones. 2016;10(3):95–106.
  25. Zhang X-J, Feng S-Y, Li Z-T, Feng Y-M. Expression of Helicobacter pylori hspA gene in Lactococcus lactis NICE system and experimental study on its immunoreactivity. Gastroent Res Pract. 2015;2015:1–6.

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